patents.google.com

CN109486766B - Lacrimal gland stem cell, culture system and culture method of lacrimal gland stem cell - Google Patents

  • ️Fri May 07 2021
Lacrimal gland stem cell, culture system and culture method of lacrimal gland stem cell Download PDF

Info

Publication number
CN109486766B
CN109486766B CN201811415998.6A CN201811415998A CN109486766B CN 109486766 B CN109486766 B CN 109486766B CN 201811415998 A CN201811415998 A CN 201811415998A CN 109486766 B CN109486766 B CN 109486766B Authority
CN
China
Prior art keywords
lacrimal gland
culture
stem cells
gland stem
cells
Prior art date
2018-11-26
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201811415998.6A
Other languages
Chinese (zh)
Other versions
CN109486766A (en
Inventor
张雁
肖洒
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sun Yat Sen University
Original Assignee
Sun Yat Sen University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
2018-11-26
Filing date
2018-11-26
Publication date
2021-05-07
2018-11-26 Application filed by Sun Yat Sen University filed Critical Sun Yat Sen University
2018-11-26 Priority to CN201811415998.6A priority Critical patent/CN109486766B/en
2019-03-19 Publication of CN109486766A publication Critical patent/CN109486766A/en
2021-05-07 Application granted granted Critical
2021-05-07 Publication of CN109486766B publication Critical patent/CN109486766B/en
Status Active legal-status Critical Current
2038-11-26 Anticipated expiration legal-status Critical

Links

  • 210000004561 lacrimal apparatus Anatomy 0.000 title claims abstract description 133
  • 210000000130 stem cell Anatomy 0.000 title claims abstract description 124
  • 238000012136 culture method Methods 0.000 title abstract description 14
  • 210000004027 cell Anatomy 0.000 claims abstract description 62
  • 108010082117 matrigel Proteins 0.000 claims abstract description 24
  • 238000004113 cell culture Methods 0.000 claims abstract description 21
  • 239000003446 ligand Substances 0.000 claims abstract description 19
  • 230000019491 signal transduction Effects 0.000 claims abstract description 14
  • 239000000654 additive Substances 0.000 claims abstract description 13
  • 239000003112 inhibitor Substances 0.000 claims abstract description 13
  • 238000000034 method Methods 0.000 claims abstract description 10
  • HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 claims abstract description 9
  • 239000001963 growth medium Substances 0.000 claims abstract description 9
  • 102000018233 Fibroblast Growth Factor Human genes 0.000 claims abstract description 8
  • 108050007372 Fibroblast Growth Factor Proteins 0.000 claims abstract description 8
  • 235000020776 essential amino acid Nutrition 0.000 claims abstract description 8
  • 239000003797 essential amino acid Substances 0.000 claims abstract description 8
  • 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 7
  • 102000007665 Extracellular Signal-Regulated MAP Kinases Human genes 0.000 claims abstract description 7
  • 108010007457 Extracellular Signal-Regulated MAP Kinases Proteins 0.000 claims abstract description 7
  • 102000043136 MAP kinase family Human genes 0.000 claims abstract description 7
  • 108090000744 Mitogen-Activated Protein Kinase Kinases Proteins 0.000 claims abstract description 6
  • 229960002648 alanylglutamine Drugs 0.000 claims abstract description 6
  • 239000011435 rock Substances 0.000 claims abstract description 6
  • 230000004156 Wnt signaling pathway Effects 0.000 claims abstract description 5
  • 102100028412 Fibroblast growth factor 10 Human genes 0.000 claims description 13
  • 101000917237 Homo sapiens Fibroblast growth factor 10 Proteins 0.000 claims description 13
  • 102000044880 Wnt3A Human genes 0.000 claims description 13
  • 108700013515 Wnt3A Proteins 0.000 claims description 13
  • 239000000203 mixture Substances 0.000 claims description 11
  • HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 10
  • CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 10
  • 230000000996 additive effect Effects 0.000 claims description 10
  • 210000001519 tissue Anatomy 0.000 claims description 9
  • 239000011159 matrix material Substances 0.000 claims description 8
  • 238000012258 culturing Methods 0.000 claims description 6
  • NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims description 6
  • ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 5
  • HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 5
  • 239000005642 Oleic acid Substances 0.000 claims description 5
  • 102000004338 Transferrin Human genes 0.000 claims description 5
  • 108090000901 Transferrin Proteins 0.000 claims description 5
  • 229930003268 Vitamin C Natural products 0.000 claims description 5
  • 239000012888 bovine serum Substances 0.000 claims description 5
  • BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims description 5
  • 229940126864 fibroblast growth factor Drugs 0.000 claims description 5
  • 229960002897 heparin Drugs 0.000 claims description 5
  • 229920000669 heparin Polymers 0.000 claims description 5
  • 229960001471 sodium selenite Drugs 0.000 claims description 5
  • 239000011781 sodium selenite Substances 0.000 claims description 5
  • 235000015921 sodium selenite Nutrition 0.000 claims description 5
  • 239000012581 transferrin Substances 0.000 claims description 5
  • 235000019154 vitamin C Nutrition 0.000 claims description 5
  • 239000011718 vitamin C Substances 0.000 claims description 5
  • DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 5
  • 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 4
  • 102000004877 Insulin Human genes 0.000 claims description 3
  • 108090001061 Insulin Proteins 0.000 claims description 3
  • 229940125396 insulin Drugs 0.000 claims description 3
  • IXIBAKNTJSCKJM-BUBXBXGNSA-N bovine insulin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 IXIBAKNTJSCKJM-BUBXBXGNSA-N 0.000 claims description 2
  • 102100024785 Fibroblast growth factor 2 Human genes 0.000 claims 1
  • 102000013814 Wnt Human genes 0.000 claims 1
  • 108050003627 Wnt Proteins 0.000 claims 1
  • 230000028327 secretion Effects 0.000 abstract description 9
  • 208000005494 xerophthalmia Diseases 0.000 abstract description 3
  • 241000699666 Mus <mouse, genus> Species 0.000 description 43
  • LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 24
  • 239000000243 solution Substances 0.000 description 19
  • 208000003556 Dry Eye Syndromes Diseases 0.000 description 14
  • 206010013774 Dry eye Diseases 0.000 description 12
  • 238000000338 in vitro Methods 0.000 description 10
  • 235000019441 ethanol Nutrition 0.000 description 8
  • 239000003102 growth factor Substances 0.000 description 8
  • XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
  • 241000699670 Mus sp. Species 0.000 description 7
  • 238000001514 detection method Methods 0.000 description 7
  • 238000002474 experimental method Methods 0.000 description 6
  • 239000012188 paraffin wax Substances 0.000 description 6
  • 230000003248 secreting effect Effects 0.000 description 6
  • 230000008859 change Effects 0.000 description 5
  • 238000010586 diagram Methods 0.000 description 5
  • 239000007788 liquid Substances 0.000 description 5
  • 230000035755 proliferation Effects 0.000 description 5
  • 210000002966 serum Anatomy 0.000 description 5
  • 238000005406 washing Methods 0.000 description 5
  • 229920000936 Agarose Polymers 0.000 description 4
  • 238000003556 assay Methods 0.000 description 4
  • 238000004140 cleaning Methods 0.000 description 4
  • 210000005036 nerve Anatomy 0.000 description 4
  • 210000000056 organ Anatomy 0.000 description 4
  • 239000002244 precipitate Substances 0.000 description 4
  • 238000002054 transplantation Methods 0.000 description 4
  • 238000011282 treatment Methods 0.000 description 4
  • QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
  • 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 3
  • HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
  • 239000003153 chemical reaction reagent Substances 0.000 description 3
  • 230000004069 differentiation Effects 0.000 description 3
  • 108010007093 dispase Proteins 0.000 description 3
  • 230000000694 effects Effects 0.000 description 3
  • 238000001962 electrophoresis Methods 0.000 description 3
  • ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 3
  • 229960005542 ethidium bromide Drugs 0.000 description 3
  • 230000014509 gene expression Effects 0.000 description 3
  • 108090000623 proteins and genes Proteins 0.000 description 3
  • 230000002829 reductive effect Effects 0.000 description 3
  • 230000008439 repair process Effects 0.000 description 3
  • 238000010839 reverse transcription Methods 0.000 description 3
  • 238000011476 stem cell transplantation Methods 0.000 description 3
  • 208000024891 symptom Diseases 0.000 description 3
  • MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
  • 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
  • NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 2
  • FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
  • 102000004392 Aquaporin 5 Human genes 0.000 description 2
  • 108090000976 Aquaporin 5 Proteins 0.000 description 2
  • 241000283707 Capra Species 0.000 description 2
  • HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
  • 229920000742 Cotton Polymers 0.000 description 2
  • KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
  • 101150040052 KRT14 gene Proteins 0.000 description 2
  • 101150023976 Krt19 gene Proteins 0.000 description 2
  • DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
  • WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
  • BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 2
  • FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
  • 229930006000 Sucrose Natural products 0.000 description 2
  • CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
  • 230000009286 beneficial effect Effects 0.000 description 2
  • 238000007664 blowing Methods 0.000 description 2
  • 239000000872 buffer Substances 0.000 description 2
  • 239000006143 cell culture medium Substances 0.000 description 2
  • 238000006243 chemical reaction Methods 0.000 description 2
  • 230000001143 conditioned effect Effects 0.000 description 2
  • 238000004043 dyeing Methods 0.000 description 2
  • 238000001976 enzyme digestion Methods 0.000 description 2
  • 238000010166 immunofluorescence Methods 0.000 description 2
  • 238000001727 in vivo Methods 0.000 description 2
  • 206010023332 keratitis Diseases 0.000 description 2
  • 230000000670 limiting effect Effects 0.000 description 2
  • 239000003550 marker Substances 0.000 description 2
  • 239000000463 material Substances 0.000 description 2
  • 238000005259 measurement Methods 0.000 description 2
  • 210000002220 organoid Anatomy 0.000 description 2
  • 210000005037 parasympathetic nerve Anatomy 0.000 description 2
  • 229960003531 phenolsulfonphthalein Drugs 0.000 description 2
  • 230000008569 process Effects 0.000 description 2
  • 230000001105 regulatory effect Effects 0.000 description 2
  • 238000011160 research Methods 0.000 description 2
  • 238000003757 reverse transcription PCR Methods 0.000 description 2
  • 238000007789 sealing Methods 0.000 description 2
  • 238000002791 soaking Methods 0.000 description 2
  • 238000003756 stirring Methods 0.000 description 2
  • 239000011550 stock solution Substances 0.000 description 2
  • 239000005720 sucrose Substances 0.000 description 2
  • 239000006228 supernatant Substances 0.000 description 2
  • 230000002889 sympathetic effect Effects 0.000 description 2
  • 238000012795 verification Methods 0.000 description 2
  • 108060003345 Adrenergic Receptor Proteins 0.000 description 1
  • 102000017910 Adrenergic receptor Human genes 0.000 description 1
  • 206010002091 Anaesthesia Diseases 0.000 description 1
  • 101100189945 Arabidopsis thaliana PER63 gene Proteins 0.000 description 1
  • 208000023275 Autoimmune disease Diseases 0.000 description 1
  • 102000000905 Cadherin Human genes 0.000 description 1
  • 108050007957 Cadherin Proteins 0.000 description 1
  • 108010009685 Cholinergic Receptors Proteins 0.000 description 1
  • 206010010741 Conjunctivitis Diseases 0.000 description 1
  • CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
  • QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
  • KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
  • ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
  • 101150084967 EPCAM gene Proteins 0.000 description 1
  • 102000004190 Enzymes Human genes 0.000 description 1
  • 108090000790 Enzymes Proteins 0.000 description 1
  • 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
  • 208000003098 Ganglion Cysts Diseases 0.000 description 1
  • DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
  • 239000007836 KH2PO4 Substances 0.000 description 1
  • 101150037996 KRT5 gene Proteins 0.000 description 1
  • QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
  • CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 1
  • ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
  • QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
  • CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
  • 229930182821 L-proline Natural products 0.000 description 1
  • 102000010445 Lactoferrin Human genes 0.000 description 1
  • 108010063045 Lactoferrin Proteins 0.000 description 1
  • 102000019298 Lipocalin Human genes 0.000 description 1
  • 108050006654 Lipocalin Proteins 0.000 description 1
  • 108091054455 MAP kinase family Proteins 0.000 description 1
  • 102000016943 Muramidase Human genes 0.000 description 1
  • 108010014251 Muramidase Proteins 0.000 description 1
  • 102000014415 Muscarinic acetylcholine receptor Human genes 0.000 description 1
  • 108050003473 Muscarinic acetylcholine receptor Proteins 0.000 description 1
  • 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
  • 102000008730 Nestin Human genes 0.000 description 1
  • 108010088225 Nestin Proteins 0.000 description 1
  • CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
  • 102000007354 PAX6 Transcription Factor Human genes 0.000 description 1
  • 101150081664 PAX6 gene Proteins 0.000 description 1
  • 229930040373 Paraformaldehyde Natural products 0.000 description 1
  • 101710098940 Pro-epidermal growth factor Proteins 0.000 description 1
  • 208000005400 Synovial Cyst Diseases 0.000 description 1
  • 101150071739 Tp63 gene Proteins 0.000 description 1
  • 239000007983 Tris buffer Substances 0.000 description 1
  • 102000004142 Trypsin Human genes 0.000 description 1
  • 108090000631 Trypsin Proteins 0.000 description 1
  • 102100027881 Tumor protein 63 Human genes 0.000 description 1
  • 208000025865 Ulcer Diseases 0.000 description 1
  • 102000012088 Vasoactive Intestinal Peptide Receptors Human genes 0.000 description 1
  • 108010075974 Vasoactive Intestinal Peptide Receptors Proteins 0.000 description 1
  • 208000027418 Wounds and injury Diseases 0.000 description 1
  • 102000034337 acetylcholine receptors Human genes 0.000 description 1
  • 230000001800 adrenalinergic effect Effects 0.000 description 1
  • 210000004504 adult stem cell Anatomy 0.000 description 1
  • 230000032683 aging Effects 0.000 description 1
  • 229960003767 alanine Drugs 0.000 description 1
  • 230000037005 anaesthesia Effects 0.000 description 1
  • 239000003098 androgen Substances 0.000 description 1
  • 238000000137 annealing Methods 0.000 description 1
  • 229940124599 anti-inflammatory drug Drugs 0.000 description 1
  • 239000000427 antigen Substances 0.000 description 1
  • 102000036639 antigens Human genes 0.000 description 1
  • 108091007433 antigens Proteins 0.000 description 1
  • 229960001230 asparagine Drugs 0.000 description 1
  • 229960005261 aspartic acid Drugs 0.000 description 1
  • 239000007640 basal medium Substances 0.000 description 1
  • 210000002469 basement membrane Anatomy 0.000 description 1
  • 230000004071 biological effect Effects 0.000 description 1
  • 230000000903 blocking effect Effects 0.000 description 1
  • 230000004663 cell proliferation Effects 0.000 description 1
  • 108091092328 cellular RNA Proteins 0.000 description 1
  • 238000005119 centrifugation Methods 0.000 description 1
  • 230000001713 cholinergic effect Effects 0.000 description 1
  • 230000001886 ciliary effect Effects 0.000 description 1
  • 230000001112 coagulating effect Effects 0.000 description 1
  • 239000002299 complementary DNA Substances 0.000 description 1
  • 210000004087 cornea Anatomy 0.000 description 1
  • 230000006378 damage Effects 0.000 description 1
  • 230000003247 decreasing effect Effects 0.000 description 1
  • 230000007547 defect Effects 0.000 description 1
  • 230000002950 deficient Effects 0.000 description 1
  • 230000029087 digestion Effects 0.000 description 1
  • BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
  • 229910000397 disodium phosphate Inorganic materials 0.000 description 1
  • 238000004090 dissolution Methods 0.000 description 1
  • 239000012153 distilled water Substances 0.000 description 1
  • 238000009826 distribution Methods 0.000 description 1
  • 229940079593 drug Drugs 0.000 description 1
  • 239000003814 drug Substances 0.000 description 1
  • 238000002651 drug therapy Methods 0.000 description 1
  • 238000001035 drying Methods 0.000 description 1
  • 230000004064 dysfunction Effects 0.000 description 1
  • 229940088598 enzyme Drugs 0.000 description 1
  • 238000000605 extraction Methods 0.000 description 1
  • 239000003889 eye drop Substances 0.000 description 1
  • 229940012356 eye drops Drugs 0.000 description 1
  • 208000027993 eye symptom Diseases 0.000 description 1
  • 238000001914 filtration Methods 0.000 description 1
  • 239000012530 fluid Substances 0.000 description 1
  • 210000000609 ganglia Anatomy 0.000 description 1
  • 210000004907 gland Anatomy 0.000 description 1
  • 230000006589 gland dysfunction Effects 0.000 description 1
  • 239000011521 glass Substances 0.000 description 1
  • 229960002989 glutamic acid Drugs 0.000 description 1
  • 238000010438 heat treatment Methods 0.000 description 1
  • 229940099472 immunoglobulin a Drugs 0.000 description 1
  • 230000028709 inflammatory response Effects 0.000 description 1
  • 230000002401 inhibitory effect Effects 0.000 description 1
  • 239000007924 injection Substances 0.000 description 1
  • 238000002347 injection Methods 0.000 description 1
  • 238000002955 isolation Methods 0.000 description 1
  • 230000002147 killing effect Effects 0.000 description 1
  • CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 1
  • 229940078795 lactoferrin Drugs 0.000 description 1
  • 235000021242 lactoferrin Nutrition 0.000 description 1
  • 230000003902 lesion Effects 0.000 description 1
  • 229960000274 lysozyme Drugs 0.000 description 1
  • 235000010335 lysozyme Nutrition 0.000 description 1
  • 239000004325 lysozyme Substances 0.000 description 1
  • 238000005360 mashing Methods 0.000 description 1
  • 239000013028 medium composition Substances 0.000 description 1
  • 108020004999 messenger RNA Proteins 0.000 description 1
  • 238000002156 mixing Methods 0.000 description 1
  • 238000012986 modification Methods 0.000 description 1
  • 230000004048 modification Effects 0.000 description 1
  • 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
  • 210000005055 nestin Anatomy 0.000 description 1
  • 230000007935 neutral effect Effects 0.000 description 1
  • 108020004707 nucleic acids Proteins 0.000 description 1
  • 102000039446 nucleic acids Human genes 0.000 description 1
  • 150000007523 nucleic acids Chemical class 0.000 description 1
  • 229940023490 ophthalmic product Drugs 0.000 description 1
  • 230000010355 oscillation Effects 0.000 description 1
  • 230000003204 osmotic effect Effects 0.000 description 1
  • 229920002866 paraformaldehyde Polymers 0.000 description 1
  • 239000011148 porous material Substances 0.000 description 1
  • 239000000843 powder Substances 0.000 description 1
  • 238000002360 preparation method Methods 0.000 description 1
  • 229960002429 proline Drugs 0.000 description 1
  • 230000002035 prolonged effect Effects 0.000 description 1
  • 238000001959 radiotherapy Methods 0.000 description 1
  • 230000000384 rearing effect Effects 0.000 description 1
  • 108091092562 ribozyme Proteins 0.000 description 1
  • 210000005077 saccule Anatomy 0.000 description 1
  • 230000001953 sensory effect Effects 0.000 description 1
  • 238000000926 separation method Methods 0.000 description 1
  • 229960001153 serine Drugs 0.000 description 1
  • 238000010008 shearing Methods 0.000 description 1
  • 239000011780 sodium chloride Substances 0.000 description 1
  • 239000007787 solid Substances 0.000 description 1
  • 238000007447 staining method Methods 0.000 description 1
  • 238000002660 stem cell treatment Methods 0.000 description 1
  • 230000001954 sterilising effect Effects 0.000 description 1
  • 210000002222 superior cervical ganglion Anatomy 0.000 description 1
  • 238000012360 testing method Methods 0.000 description 1
  • 230000001225 therapeutic effect Effects 0.000 description 1
  • 238000002560 therapeutic procedure Methods 0.000 description 1
  • 210000003901 trigeminal nerve Anatomy 0.000 description 1
  • LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
  • 239000012588 trypsin Substances 0.000 description 1
  • 231100000397 ulcer Toxicity 0.000 description 1
  • 229910021642 ultra pure water Inorganic materials 0.000 description 1
  • 239000012498 ultrapure water Substances 0.000 description 1
  • 238000012418 validation experiment Methods 0.000 description 1
  • 238000003260 vortexing Methods 0.000 description 1
  • 238000005303 weighing Methods 0.000 description 1
  • 239000008096 xylene Substances 0.000 description 1

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/44—Thiols, e.g. mercaptoethanol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/405—Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
    • C12N2501/91—Heparin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Neurology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Neurosurgery (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Materials For Medical Uses (AREA)

Abstract

The invention relates to a lacrimal gland stem cell, a culture system and a culture method of the lacrimal gland stem cell, and relates to the field of cell engineering. The culture system of the lacrimal gland stem cells takes DMEM/F12 as a basic culture medium, and the following components are added to form a culture solution: cell culture additives, non-essential amino acids, L-alanyl-L-glutamine, mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway ligands, fibroblast growth factors, Wnt signaling pathway ligands and ROCK signaling pathway inhibitors, and matrigel as a scaffold for stereoculture. The culture method of the invention is that mouse lacrimal gland tissue is digested into single cells and inoculated into matrigel, and after the single cells are solidified, the single cells are added into the lacrimal gland stem cell culture solution for primary culture. The mouse lacrimal gland stem cells cultured by the method are large in quantity, can be stably and continuously passed, have the function of restoring the lacrimal gland secretion function, and can effectively treat xerophthalmia.

Description

Lacrimal gland stem cell, culture system and culture method of lacrimal gland stem cell

Technical Field

The invention relates to the field of cell engineering, in particular to a three-dimensional culture system and a mouse Lacrimal Gland Stem Cell (LGSC) cultured by adopting the culture system.

Background

The lacrimal gland is an important organ of the eye and is mainly composed of secretory acini and secretory canals. The lacrimal gland is regulated by sympathetic and parasympathetic nerves, most of which are cholinergic nerves and a small of which are adrenergic nerves. Sympathetic nerves originate from the superior cervical ganglia, parasympathetic nerves connect with the pterygopalatine ganglion and ciliary ganglia, and certain sensory nerves of the lacrimal gland connect with the trigeminal nerve. There are a number of acetylcholine receptors in the lacrimal gland, including muscarinic receptors, vasoactive intestinal peptide receptors type I and II, and

norepinephrine receptors α

1, β. The neurally regulated lower lacrimal gland can secrete lysozyme, lactoferrin, lipocalin, immunoglobulin A and the like.

The lacrimal gland secretes tears to form a tear film, which keeps the eye moist to protect the eye. Once the lacrimal gland is diseased or injured, the tear secretion is deficient and the person suffers from Dry Eye Disease (DED). Lacrimal gland dysfunction is caused by aging, autoimmune disorders, radiation therapy of the eye, and decreased androgen levels in humans. The tear film secreted and formed by the patient with dry eye is higher than the normal tear film osmotic pressure, so that corneal inflammation is caused, corneal cells are damaged, ulcer is formed, the vision of the patient is finally reduced, and the life quality of the patient is seriously influenced.

For patients with dry eye, the current drug treatment scheme mainly comprises dripping isotonic or hypotonic eye drops, inhibiting the inflammation of conjunctiva and cornea of eyes caused by dry eye, blocking lacrimal pores and increasing the thickness of lacrimal film, and the like. However, these treatments only alleviate the symptoms of dry eye, keep the eye as moist as possible, and avoid damage to the corneal cells. Furthermore, prolonged use of ophthalmic drugs and anti-inflammatory drugs also kills corneal cells and triggers a more refractory inflammatory response. Therefore, the drug therapy cannot fundamentally solve the problem of lacrimal gland secretory dysfunction, and cannot completely cure aged or diseased lacrimal glands.

In order to treat dry eye more effectively and thoroughly, researchers have conducted a series of studies aimed at restoring the secretory function of the lacrimal gland. To fully restore the secretory function of the lacrimal gland, new healthy cells or organs are needed for transplantation therapy. In order to obtain lacrimal cells for transplantation, researchers have begun attempting to culture lacrimal stem cells by in vitro isolation. Through recent thirty years of efforts, the in vitro culture of the lacrimal gland stem cells has breakthrough research, however, the prior in vitro culture method of the lacrimal gland stem cells has very low efficiency and small quantity of sorting progenitor cells, can not be continuously subcultured, and greatly limits the clinical application of the lacrimal gland stem cells. Therefore, a better separation method and culture system are needed to separate and culture lacrimal gland stem cells, and a good technical platform is provided for clinical application.

Disclosure of Invention

In view of the above, it is necessary to provide a culture system and a culture method for lacrimal gland stem cells, which can obtain a large number of lacrimal gland stem cells and can perform stable subculture, aiming at the problems of low efficiency and small number of progenitor cell sorting in the existing culture system and culture method for lacrimal gland stem cells.

A culture system of lacrimal gland stem cells takes DMEM/F12 as a basal medium, and the following components are added to form a culture solution: cell culture additives, non-essential amino acids, L-alanyl-L-glutamine, mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway ligands, fibroblast growth factors, Wnt signaling pathway ligands and ROCK signaling pathway inhibitors, and matrigel as a scaffold for stereoculture.

Further, the cell culture additive is a mixture of N2 and B27.

The cell culture additive can also be 9F, and comprises beta-mercaptoethanol, 2-aminoethanol, sodium selenite, heparin, transferrin, vitamin C, insulin, bovine serum albumin-oleic acid and fibroblast growth factor-2 (FGF 2).

Further, the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway (MAPK/ERK signaling pathway) ligand is EGF, the fibroblast growth factor is FGF10, the Wnt signaling pathway ligand is Wnt3A, and the ROCK signaling pathway inhibitor is Y-27632.

Further, the concentration of N2 in the cell culture additive is 1%, and the concentration of B27 in the cell culture additive is 2%; the concentration of the non-essential amino acid is 1 percent, and the concentration of the L-alanyl-L-glutamine is 1 percent.

Further, the cell culture additive 9F comprises the following components in concentration: 5-10 mu M of beta-mercaptoethanol, 5-10 mu M of 2-aminoethanol, 10-20 mu M of sodium selenite, 50-150ng/ml of heparin, 2-5ng/ml of transferrin, 5-10ng/ml of vitamin C, 5-10ng/ml of insulin, 6.8-9.4ng/ml of bovine serum albumin-oleic acid and-25-10 ng/ml of fibroblast growth factor; 1% of said non-essential amino acids, 1% of said L-alanyl-L-glutamine.

Further, the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway ligand is EGF, the fibroblast growth factor is FGF10, the Wnt signaling pathway ligand is Wnt3A, and the ROCK signaling pathway inhibitor is Y-27632.

Furthermore, the concentration of EGF in the cell culture additive is 10-50ng/ml, the concentration of FGF10 is 10-100ng/ml, the concentration of Wnt3A is 5-10ng/ml, and the concentration of Y-27632 is 5-10 mM.

Further, the Matrigel is Matrigel. The Matrigel is a three-dimensional matrix with biological activity, simulates the structure, composition, physical characteristics and functions of an in-vivo cell basement membrane, and is adopted as Matrigel to be beneficial to culture and differentiation of mouse lacrimal gland stem cells cultured in vitro.

Furthermore, the non-essential amino acid uses NEAA, which contains 7 non-essential amino acids such as Gly, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, etc., and can effectively improve the proportion of the cell culture medium.

A process for culturing the lacrimal gland stem cell includes such steps as digesting the tissue of mouse lacrimal gland to become single cells, inoculating it to matrix gel, coagulating, adding it to the culture liquid of said lacrimal gland stem cell, culturing at 37 deg.C and 5-10% CO2And performing primary culture under the environment.

A lacrimal gland stem cell is prepared by the culture system and the culture method.

Compared with the prior art, the invention has the following beneficial effects:

the invention overcomes the defect that the traditional plane culture method can not effectively culture to obtain the lacrimal gland stem cells, constructs a three-dimensional culture method to culture the mouse lacrimal gland stem cells in vitro, and the mouse lacrimal gland stem cells cultured by the method have more quantity, thereby providing sufficient cell materials for clinical research;

the lacrimal gland stem cells are cultured in a three-dimensional way by using matrigel, the stem cells can be self-replicated along with the time lapse, and one part of the stem cells can be differentiated to form a micro organ similar to the organ structure or the cell updating process, namely an 'organoid body', the stem cells interact with the differentiated cells in the organoid body, and the signal molecules interact with each other to form a relatively stable stem cell proliferation microenvironment, so that the stem cells can be continuously passaged and certain tissue characteristics and genotype stability are maintained;

the mouse lacrimal gland stem cell obtained by the in-vitro culture system established by the invention has the advantages that the secretion of transplanted mouse lacrimal fluid is obviously increased, the damaged lacrimal gland is repaired, the function of restoring the lacrimal gland secretion is realized, and a good foundation is provided for researching stem cell treatment of xerophthalmia.

Drawings

Fig. 1 is a state change diagram of different culture generations when the mouse lacrimal gland stem cells are continuously passaged under the condition of LGSCM, and the scale bar: 100 μm;

FIG. 2 is a statistical population of cells from different culture generations of LGSCM conditioned mouse lacrimal gland stem cells following continuous passage;

fig. 3 is a graph of the change in status of LGSCM conditioned mouse lacrimal gland stem cells with increasing culture time, scale: 100 μm;

FIG. 4 is a state diagram of primary cultured lacrimal stem cells, a, LGSCM, b, 9F-LGSCM, scale: 100 μm;

FIG. 5 is a diagram of RT-PCR detection of the expression of sternness genes of lacrimal gland stem cells of different generations;

fig. 6 is a state diagram of primary cultured lacrimal stem cells (P0 generation) with the removal of each growth factor, ligand, and inhibitor alone at

day

7, scale: 400 μm;

figure 7 is a graph of the size and total number of cells after 7 days of culture with the individual growth factors, ligands and inhibitor P0 removed alone, P < 0.01;

fig. 8 is a state diagram of lacrimal gland stem cell withdrawal with P2 as the growth factor, ligand and inhibitor alone at

day

7, scale: 400 μm;

figure 9 is the size and total number of cells after 7 days of culture with the individual growth factors, ligands and inhibitor P2 removed alone, P < 0.01;

fig. 10 is an IF assay of lacrimal stem cells cultured for 14 days, scale: 100 μm;

fig. 11 is an IF detection map of the lacrimal gland of a mature mouse, scale bar: 100 μm;

fig. 12 is an IF assay of lacrimal stem cells cultured for more than 14 days, scale bar: 200 mu m;

FIG. 13 is a graph of ocular symptoms before and after treatment in a mouse with dry eye;

fig. 14 is an IHC assay of a section of lacrimal gland 8 weeks after transplantation of lacrimal gland stem cells in a mouse with dry eye syndrome, scale: 100 μm;

fig. 15 is the amount of tear secretion in dry eye mice transplanted with lacrimal gland stem cells for 8 weeks, P < 0.05; p < 0.01.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Description of materials:

in the experiment, the mouse is purchased from the animal center of medical experiment in Guangdong province; n2, B27, Glutamax, NEAA were purchased from Gibco, EGF, FGF10, Wnt3A were purchased from Pepro Tech, Y-27632 was purchased from Sigma.

The main solution and the formula are as follows:

(1) solutions or reagents for use in cell culture experiments

1) PBS Buffer configuration: weighing 8g of NaCl, 0.2g of KCl and Na2HPO4 1.155g，KH2PO40.2g, adding about 800ml of ultrapure water, fully dissolving, adjusting the pH value to 7.4, fixing the volume to 1L, sterilizing at high temperature and high pressure, and storing at 4 ℃.

2) Preparation of 10 XTrypsin-EDTA (TE) 1.25g of Trypsin and 1.00g of EDTA are weighed and dissolved in 250ml of PBS, filtered and sterilized, and stored at-20 ℃. Cells were digested with PBS diluted to 1 XTE prior to use.

3) When neutral enzyme (Dispase) was purchased from BD as it was for primary culture, lacrimal gland tissue was digested and used.

4) Lacrimal gland stem cell culture solution: LGSCM, 9F-LGSCM.

5) The DMEM/F12(Sigma) medium composition is shown in the following table:

Figure BDA0001879476330000041

Figure BDA0001879476330000051

(2) solutions or reagents for electrophoresis of nucleic acids

1)50 XTAE electrophoresis Buffer (pH8.5): into a 1L glass bottle was added 242g of Tris, 18.6g of Na2EDTA·2H2And O, adding about 800ml of secondary water, fully stirring for dissolving, adding 57.1ml of acetic acid, fully stirring, adding the secondary water to a constant volume of 1L, and storing at room temperature. It is diluted 50 times when used.

2) Ethidium Bromide (EB): the stock solution concentration was 10mg/ml and the working concentration was 0.5. mu.g/ml.

3) 1% agarose electrophoresis gel: 1g of agarose (agarose) was weighed, 100ml of 1 XTAE buffer was added, the mixture was heated and boiled until the agarose was completely dissolved, cooled to about 50 ℃, 5. mu.l of EB stock solution was added to a final concentration of 0.5. mu.g/ml, and the mixture was shaken well and poured onto a gel plate.

(3) Solutions or reagents for use in immunofluorescence experiments

1) 4% PFA: 20g of paraformaldehyde were dissolved in 400ml of 1 XPBS and dissolved overnight (about 12 hours) at 65 ℃ with heating. When a small amount of precipitate remained at the bottom, 40 μ l of 1N NaOH was added to promote dissolution, and the volume was determined to be 500ml, and the mixture was stored at 4 ℃.

2) 30% sucrose solution: 3g of sucrose powder was dissolved in 4% PFA, and the solution was taken up to a volume of 10ml and stored at 4 ℃.

3) Sealing liquid: the nonimmune goat serum was diluted to 10% with 1 × PBS and stored as prepared.

4) A first antibody: krt14(Abcam, ab181595), Krt19(Abcam, ab52625), AQP5(Abcam, ab104751), E-cadherin (Abcam, ab11512), mCherry (Abcam, ab167453)

5) Secondary antibody: purchased from life corporation.

Example 1:

a culture system of lacrimal gland stem cells takes DMEM/F12 as a basic culture medium, and the following components and concentrations are added to form a culture solution: 1% N2, 2% B27, 1% NEAA, 1% Glutamax, 10-50ng/ml EGF, 10-100ng/ml FGF10, 5-10ng/ml Wnt3A, 5-10mM Y-27632, and Matrigel as a scaffold for three-dimensional culture. The culture broth is referred to herein as LGSCM.

Example 2:

a culture system of lacrimal gland stem cells takes DMEM/F12 as a basic culture medium, and the following components and concentrations are added to form a culture solution: 5-10 MuM beta-mercaptoethanol, 5-10 MuM 2-aminoethanol, 10-20nM sodium selenite, 50-150ng/ml heparin, 2-5ng/ml transferrin, 5-10ng/ml vitamin C, 5-10 mug/ml insulin, 6.8-9.4 mug/ml bovine serum albumin-oleic acid, 5-10ng/ml FGF2, 1% NEAA, 1% Glutamax, 10-50ng/ml EGF, 10-100ng/ml FGF10, 5-10ng/ml Wnt3A, 5-10mM Y-27632, and Matrigel as a three-dimensional culture scaffold. The culture medium is referred to herein as 9F-LGSCM

Example 3:

a culture method of lacrimal gland stem cells comprises the following steps:

a) separating by adopting an enzyme digestion method to obtain mouse lacrimal gland single cells;

b) according to 1 × 104The ratio of cells/holes is planted into Matrigel matrix, and added into prepared lacrimal gland stem cell culture solution LGSCM for culture, the culture environment is 37 ℃, and 5-10% CO2After 7 days of culture, a saccular stem cell population was formed, and 1X 10 cells were used4Cell/well ratio passaging.

Experimental example 4:

a culture method of lacrimal gland stem cells comprises the following steps:

a) separating by adopting an enzyme digestion method to obtain mouse lacrimal gland single cells;

b) according to 1 × 104The ratio of cells/holes is planted into Matrigel matrix, and added into the prepared lacrimal gland stem cell culture solution 9F-LGSCM for culture under the culture environment of 5-10% CO at 37 DEG C2After 7 days of culture, a saccular stem cell population was formed, and 1X 10 cells were used4Cell/well ratio passaging.

Example 5:

lacrimal gland stem cells obtained by the culture method of example 3 or example 4 were used.

In order to support the above embodiments, the present invention performs a series of experiments such as primary culture and subculture of mouse lacrimal gland stem cells, composition verification of a culture solution of lacrimal gland stem cells, identification of mouse lacrimal gland stem cells, and treatment of dry eye of mouse lacrimal gland stem cells, and the specific experimental steps of each experiment are as follows:

experimental example 1: primary culture of mouse lacrimal gland stem cells

1) Killing the mouse, dissecting to obtain a lacrimal gland of the mouse, soaking and cleaning the lacrimal gland for 1 minute by 1 multiplied by PBS, soaking the lacrimal gland for 15 seconds by 75 percent ethanol, and immediately cleaning the lacrimal gland for 2 times by 1 multiplied by PBS;

2) shearing the tissues as much as possible, transferring the cut tissues into a 15ml centrifuge tube, and adding 500 mu l of Dispase for digestion at 37 ℃ for 1 h;

3) blowing the digested tissue with a 1ml micropipette gun to disperse the tissue to obtain single cells, and washing twice with pre-cooled 1 × PBS;

4) the cells were collected after filtration through a 40 μm filter, counted and counted at 1X 104The cell/well ratio was resuspended in 40. mu.l of culture medium, mixed with 40. mu.l of Matrigel, seeded into a 24-well plate, placed in an incubator at 37 ℃ to coagulate Matrigel, and cultured after 20 minutes by adding a prepared stem cell culture medium.

The state change of primary and continuous passage different culture generations under the condition of mouse lacrimal gland stem cells LGSCM is shown in figure 1; the state of the primary cultured lacrimal gland stem cells is shown in figure 4, wherein a is LGSCM as a culture solution, b is 9F-LGSCM as a culture solution, and the diameter of the lacrimal gland stem cell mass cultured by the 9F-LGSCM is slightly smaller than that of the lacrimal gland stem cell mass cultured by the LGSCM.

Experimental example 2: lacrimal gland stem cell subculture

1) Adding 1 XPBS and 20

ul Dispase

100 ul per well in 24-well plate, mashing Matrigel and cell mixture, digesting in 37 deg.C incubator for 30 min, digesting Matrigel into liquid form,

2) collecting the cultured lacrimal gland stem cells by using a 15ml centrifuge tube, centrifuging for 4 minutes at 500rpm, and collecting precipitates;

3) adding 1 XTE digested cells, and incubating at 37 ℃ for 10 minutes;

4) terminating TE, blowing the lacrimal gland stem cells into single cells, centrifuging at 1200rpm for 4 minutes, collecting cells, and performing 1 × 104Cell/well ratioSuspended in 40. mu.l of culture medium, mixed with 40. mu.l of Matrigel, seeded in a 24-well plate, placed in an incubator at 37 ℃ to coagulate Matrigel, and cultured 20 minutes later with LGSCM or 9F-LGSCM, which is a pre-prepared culture medium for stem cells.

The mouse lacrimal gland stem cells cultured by the method are stably passed to more than 40 generations under the condition of stem cell culture solution, and the continuous passage culture time is more than 1 year. The state change of the mouse lacrimal gland stem cells after continuous passage and different culture generations is shown in figure 1; statistics of the total number of cells of the mouse lacrimal gland stem cells cultured for 7 days in different continuous subculture generations are shown in fig. 2; the change of state of mouse lacrimal gland stem cells with the increase of culture time is shown in fig. 3.

And (4) analyzing results: the culture of the mouse lacrimal gland stem cell is generally 7 days, the mouse lacrimal gland stem cell shows bud branching in primary culture, the shape of the mouse lacrimal gland stem cell is kept in a solid sacculus shape after passage, and the state is relatively stable along with the increase of the passage frequency, as shown in figure 1. The statistics of the total number of cells of different generations shows that the lacrimal gland stem cells have stronger proliferation capacity under the condition of LGSCM (LGSCM), namely 1 multiplied by 104After 7 days of culture, 4X 10 cells can be expanded5More than one cell. And with the increase of the passage times, the lacrimal gland stem cells gradually adapt to the environment of in vitro culture, the proliferation capacity of the lacrimal gland stem cells is gradually enhanced, and finally the lacrimal gland stem cells tend to be stable, as shown in figure 2. The state of lacrimal gland stem cells is changed under LGSCM conditions at different culture time, and the formed saccule cell mass is larger and larger, as shown in figure 3. Similarly, the 9F-LGSCM condition also yielded similar saccular lacrimal stem cells, which also had properties similar to those of LGSCM condition, as shown in fig. 4. Since the proliferation of lacrimal gland stem cells is relatively slow under 9F-LGSCM conditions, we used lacrimal gland stem cells under LGSCM conditions in various validation experiments that follow.

Experimental example 3: verification of components of lacrimal gland stem cell culture solution

In order to verify the necessity of the major growth factors, ligands, and inhibitors in the culture medium, the growth factors, ligands, and inhibitors were individually removed by the culture methods of experimental examples 1 and 2, respectively, and corresponding culture solutions were prepared, and the growth state and proliferation rate of lacrimal gland stem cells were individually cultured and compared.

Wnt3A, FGF10, Y-27632 and EGF were withdrawn separately, and the state of primary cultured lacrimal gland stem cells (P0 passage) at

day

7 is shown in FIG. 6;

the sizes and the total number of the cells after the culture of the lacrimal gland stem cells with Wnt3A, FGF10, Y-27632 and EGF and P0 independently removed and cultured for 7 days are shown in figure 7;

the state of

day

7 of Wnt3A, FGF10, and P2-substituted lacrimal stem cells alone, respectively, is shown in fig. 8;

the sizes and total number of cells after 7 days of culture of Wnt3A, FGF10, and P2-substituted lacrimal stem cells, respectively, were withdrawn individually, are shown in fig. 9.

And (4) analyzing results: after removing each of the major growth factors in LGSCM, we found that removal of EGF, Y-27632, FGF10 had a significant effect on primary culture of lacrimal stem cells, with a significant decrease in the balloon diameter and total cell number of lacrimal stem cells. Withdrawal of Wnt3A also had a significant effect after 2 passages. Therefore, it can be speculated that each growth factor, ligand and inhibitor added in the culture system established by the inventor plays an important role in the growth and proliferation of the lacrimal gland stem cells.

Experimental example 4: detection of tear gland stem cell gene expression mRNA level

i. Extraction of cellular RNA

1) Collecting cells cultured for 14 days in different generations and cultured for different days in the same generation, adding 1ml of Trizol, and performing vortex oscillation until the cells are completely lysed;

2) adding 0.2ml chloroform, vortexing and shaking for 30 seconds, standing at room temperature for 10 minutes until the liquid is layered, and centrifuging at 12000rpm and 4 ℃ for 15 minutes;

3) after centrifugation, the liquid in the centrifuge tube can be cleared and observed to be divided into three layers, the transparent water phase at the uppermost layer is carefully absorbed and transferred into a new 1.5ml RNase-free centrifuge tube;

4) adding isopropanol with the same volume, gently mixing, standing at room temperature for 10 minutes, and centrifuging at 12000rpm4 ℃ for 15 minutes;

5) discarding the supernatant, adding 1ml of 75% ethanol, reversing and cleaning, centrifuging at 12000rpm and 4 ℃ for 5 minutes, and discarding the supernatant;

6) placing the centrifuge tube in an ultraclean workbench, drying, and adding 20 μ l of non-RNA enzyme water to dissolve the precipitate when the white precipitate becomes semitransparent;

RT-PCR reverse transcription reaction (TOYOBO ReverTra Ace kit)

1) Adding the following components in sequence into a PCR reaction tube:

total RNA 2. mu.l (1000ng)

Acting at 65 ℃ for 5 minutes;

2) after 2 minutes ice bath of denatured RNA, add:

Figure BDA0001879476330000091

acting at 37 ℃ for 5 minutes;

3) placing on ice, and adding 5 × RT Master Mix II;

4) the reverse transcription procedure was 15 minutes at 37 ℃, 5 minutes at 50 ℃, 5 minutes at 98 ℃ and 5 minutes at 4 ℃ in an ice bath, and the cDNA sample after completion of reverse transcription was stored at-20 ℃ for further use.

PCR procedure

1) In a total volume of 20. mu.l of PCR system:

Figure BDA0001879476330000092

2) different primers are set according to various annealing temperatures, cycle numbers, extension times and the like;

3) and (5) detecting the result of nucleic acid electrophoresis.

And (4) analyzing results: as a result of the nucleic acid electrophoresis examination, as shown in FIG. 5, the cultured lacrimal gland stem cells expressed the marker genes of adult stem cells such as Epcam, Krt5, Krt14, Pax6, P63, and Nestin, and the marker genes of the individual stem cells were stably expressed by examining the lacrimal gland stem cells cultured in different generations.

Experimental example 5: mouse lacrimal gland stem cell in vitro differentiation and mouse lacrimal gland IF detection

1) The mouse lacrimal gland stem cells obtained by culture are cultured in vitro for 14 days and added with serum or the hardness of matrigel is reduced to lead the differentiation to appear,

2) fixing the differentiated lacrimal gland stem cells or mouse lacrimal gland 4% PFA solution overnight;

3) and (3) dehydrating: 70% ethanol for 2 hours, 80% ethanol for 2 hours, 95% ethanol for 30 minutes, absolute ethanol for 2 hours, absolute ethanol + TO (1:1) for 30 minutes, TO for 2 hours, TO + paraffin (1:1) for 30 minutes, paraffin for 3 hours, paraffin for 2 hours. 12 barrels in total and 16 hours;

4) embedding paraffin;

5) slicing paraffin, wherein the thickness of the slices is 4 mu m;

6) the dyeing steps are as follows:

slicing paraffin for 3 minutes at 60 ℃, carrying out xylene for 10 minutes, carrying out absolute ethyl alcohol for 3 minutes, carrying out 90% ethyl alcohol for 3 minutes, carrying out 80% ethyl alcohol for 3 minutes, carrying out 70% ethyl alcohol for 3 minutes, and carrying out distilled water washing for 3 times and 3 minutes each time;

repairing antigen for 17 min;

③ washing 3 times with 1 XPBS on a shaking bed, 5 minutes each time;

fourthly, after sealing the goat serum with 10 percent for 30 minutes, adding primary antibody, and incubating overnight at 4 ℃;

removing primary antibody, washing with 1 × PBS for 3 times, each time for 5 minutes;

sixthly, adding the secondary antibody, and incubating for 1 hour at room temperature;

removing the secondary antibody, and washing with 1 × PBS for 3 times, 5 minutes each time;

adding DAPI for dyeing at room temperature for 5 minutes;

ninthly, removing DAPI, and cleaning with 1 × PBS for 3 times, 5 minutes each time;

the red spot was encapsulated with fluorescent encapsulating tablets and photographed.

The results of IF detection of lacrimal gland stem cells cultured for 14 days with reduced matrigel hardness and the results of IF detection of lacrimal gland stem cells cultured for 14 days with serum are shown in FIG. 10 and FIG. 12, respectively. The results of IF measurements on lacrimal tissues are shown in FIG. 11. The lacrimal gland stem cells in fig. 10a and 11a show red fluorescence, and the mature luminal cells in fig. 10b and 11b show red fluorescence. In FIG. 12, a shows green fluorescence for acinar-like cells, b shows red fluorescence for lacrimal stem cells, and c shows blue fluorescence for nuclei.

And (4) analyzing results: immunofluorescence detection shows that only part of cells in a lacrimal gland stem cell mass cultured for 14 days express Krt14 and are mainly concentrated on the outer layer, which indicates that the cells are not differentiated and can maintain the characteristics of the stem cells; while another part of the cells expressed Krt19, mainly concentrated on the inside, indicating that the part of the cells had differentiated into cells with luminal property. Comparing the location expression of the two types of cells in the lachrymal tissue lumen, the distribution location of the two types of cells was found to be similar to the location in the lachrymal lumen. As can be seen from FIG. 12, the lacrimal gland stem cells cultured for 14 days by adding the serum also differentiate to express AQP5, and acinar-like cells with low nucleoplasm ratio are obtained, which indicates that the lacrimal gland stem cells obtained by culturing have the potential of differentiating to lumen and acinus in vitro culture.

Experimental example 6: in vivo repair of damaged lacrimal gland by lacrimal gland stem cells

i. Lacrimal gland stem cell transplantation

1) Culturing lacrimal gland stem cells from mice with red fluorescence (td-Tomato) to the seventh day;

2) the lacrimal gland stem cells were digested into single cells, resuspended in matrix of equal mix of Matrigel and DMEM/F12 to make the cells 2X 106concentration of cells/ml;

3) after anesthesia of a lacrimal gland injured xerophthalmia model mouse, the lacrimal gland is dissected and exposed;

4) 20000 cells were injected on the left side and resuspended cell matrix was injected on the right side as control;

5) after injection, the wound was sutured and after 8 weeks of rearing, the repairing effect was examined.

Testing the therapeutic effect after stem cell transplantation

1) Anaesthetizing the mouse and shooting eye symptoms of the mouse;

2) measuring the amount of lacrimal secretion of the mouse by using a homemade phenol red cotton thread, placing the phenol red cotton thread in the canthus of the mouse for about 10 seconds, and then measuring the length of the discolored part;

3) the mice were dissected, lacrimal glands removed, fixed, sectioned and subjected to IHC assay. The sectioning and staining methods were the same as in experimental example 5.

For example, as shown in fig. 13, IHC measurement of a tear section of a dry eye mouse transplanted with a tear stem cell for 8 weeks is shown in fig. 14, and tear secretion of a dry eye mouse transplanted with a tear stem cell for 8 weeks is shown in fig. 15.

And (4) analyzing results: after the lacrimal gland stem cells were injected, the eye dry eye symptoms of the mice were suppressed and alleviated (left side of fig. 13), while the eyes of the mice on the side of the injected matrix were significantly severely ulcerated (right side of fig. 13); after the mouse is dissected, lacrimal gland stem cells derived from transplantation can be detected in a lacrimal gland section, and are differentiated into acinar cells and lumen cells of the lacrimal gland (on the left side of figure 14), while differentiated new acinar cells and lumen cells are not found in a control group (on the right side of figure 14), which shows that the lacrimal gland stem cells have the capacity of differentiating in the mouse body and can repair damaged lacrimal glands. Compared with the lacrimal secretion of Normal (Normal), lesion treated (LGSC) and control (Vehicle) mice (fig. 15), the lacrimal gland stem cell transplanted mice were slightly lower than Normal, but significantly increased in lacrimal secretion compared to the non-transplanted stem cell group, indicating that the lacrimal gland stem cell transplantation not only can repair the damaged lacrimal gland, but also has the function of recovering the secretory function.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (3)

1. A culture system of lacrimal gland stem cells is characterized in that DMEM/F12 is used as a basic culture medium, and the following components are added to form a culture solution: a composition of cell culture additives, non-essential amino acids, L-alanyl-L-glutamine, mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway ligands, fibroblast growth factors, Wnt signaling pathway ligands and ROCK signaling pathway inhibitors, and matrigel as a scaffold for three-dimensional culture;

the cell culture additive is a mixture of N2 and B27; or the cell culture additive is 9F, and the 9F is a mixture of beta-mercaptoethanol, 2-aminoethanol, sodium selenite, heparin, transferrin, vitamin C, insulin, bovine serum albumin-oleic acid and fibroblast growth factor-2;

the mitogen-activated protein kinase/extracellular signal-regulated kinase signal pathway ligand is EGF, the fibroblast growth factor is FGF10, the Wnt signal pathway ligand is Wnt3A, and the ROCK signal pathway inhibitor is Y-27632;

the volume concentration of N2 in the cell culture additive is 0.5-1%, and the volume concentration of B27 is 1-2%;

the cell culture additive 9F comprises the following components in concentration: 5-10 mu M of beta-mercaptoethanol, 5-10 mu M of 2-aminoethanol, 10-20 mu M of sodium selenite, 50-150ng/ml of heparin, 2-5ng/ml of transferrin, 5-10ng/ml of vitamin C, 5-10ng/ml of insulin, 6.8-9.4ng/ml of bovine serum albumin-oleic acid and-25-10 ng/ml of fibroblast growth factor;

the concentration of the non-essential amino acid is 1 percent, and the concentration of the L-alanyl-L-glutamine is 1 percent;

the concentration of EGF is 10-50ng/ml, the concentration of FGF10 is 10-100ng/ml, the concentration of Wnt3A is 5-10ng/ml, and the concentration of Y-27632 is 5-10 mM.

2. The culture system of lacrimal stem cells of claim 1, wherein the Matrigel is Matrigel.

3. A method for culturing the stem cells of lacrimal gland includes such steps as digesting the tissue of lacrimal gland of mouse to become single cells, inoculating it to matrix gel, coagulatingAfter fixation, adding into the lacrimal gland stem cell culture solution of any of claims 1-2, at 37 deg.C, 5% -10% CO2Culturing is carried out under the environment.

CN201811415998.6A 2018-11-26 2018-11-26 Lacrimal gland stem cell, culture system and culture method of lacrimal gland stem cell Active CN109486766B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811415998.6A CN109486766B (en) 2018-11-26 2018-11-26 Lacrimal gland stem cell, culture system and culture method of lacrimal gland stem cell

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811415998.6A CN109486766B (en) 2018-11-26 2018-11-26 Lacrimal gland stem cell, culture system and culture method of lacrimal gland stem cell

Publications (2)

Publication Number Publication Date
CN109486766A CN109486766A (en) 2019-03-19
CN109486766B true CN109486766B (en) 2021-05-07

Family

ID=65696705

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811415998.6A Active CN109486766B (en) 2018-11-26 2018-11-26 Lacrimal gland stem cell, culture system and culture method of lacrimal gland stem cell

Country Status (1)

Country Link
CN (1) CN109486766B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110628695A (en) * 2019-11-12 2019-12-31 安徽科门生物科技有限公司 Culture medium for stem cell amplification
CN113444679B (en) * 2021-06-27 2023-12-15 深圳市眼科医院 Human lacrimal gland stem cells and differentiation culture method and application thereof
KR102547900B1 (en) * 2022-02-18 2023-06-26 차의과학대학교 산학협력단 Preparation method of lacrimal gland and use thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0613756D0 (en) * 2006-07-12 2006-08-23 Univ Sheffield Cell culture medium
ES2970720T3 (en) * 2013-03-15 2024-05-30 Astellas Inst For Regenerative Medicine Photoreceptors and photoreceptor progenitors produced from pluripotent stem cells
JP7367951B2 (en) * 2016-05-18 2023-10-24 慶應義塾 Cell culture medium for organoid culture

Also Published As

Publication number Publication date
CN109486766A (en) 2019-03-19

Similar Documents

Publication Publication Date Title
JP5395058B2 (en) 2014-01-22 Retinal pigment epithelial cells derived from stem cells
JP5391488B2 (en) 2014-01-15 RNAi methods and compositions for stimulating proliferation of cells with adhesive bonds
Cejka et al. 2016 [Retracted] The Favorable Effect of Mesenchymal Stem Cell Treatment on the Antioxidant Protective Mechanism in the Corneal Epithelium and Renewal of Corneal Optical Properties Changed after Alkali Burns
US20140370007A1 (en) 2014-12-18 Method of directed differentiation producing corneal endothelial cells, compositions thereof, and uses thereof
CN109486766B (en) 2021-05-07 Lacrimal gland stem cell, culture system and culture method of lacrimal gland stem cell
CN104946591A (en) 2015-09-30 Improved methods of producing RPE cells and compositions of RPE cells
CN105838676A (en) 2016-08-10 Culture solution for retinal pigment epitheliums and preparation method and application thereof
Lin et al. 2019 Three dimensional culture of potential epithelial progenitor cells in human lacrimal gland
Maqueda et al. 2021 Repopulation of decellularized retinas with hiPSC-derived retinal pigment epithelial and ocular progenitor cells shows cell engraftment, organization and differentiation
US20210308187A1 (en) 2021-10-07 Modalities for the treatment of degenerative diseases of the retina
Lyu et al. 2020 Protein kinase A inhibitor H89 attenuates experimental proliferative vitreoretinopathy
Peng et al. 2014 Survival and migration of pre-induced adult human peripheral blood mononuclear cells in retinal degeneration slow (rds) mice three months after subretinal transplantation
Davari et al. 2013 Amniotic fluid promotes the appearance of neural retinal progenitors and neurons in human RPE cell cultures
Ma et al. 2019 Bone marrow mesenchymal stem cells modified by angiogenin‐1 promotes tissue repair in mice with oxygen‐induced retinopathy of prematurity by promoting retinal stem cell proliferation and differentiation
CN115531297B (en) 2024-09-17 Injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell spheres, and preparation method and application thereof
EP1812560A1 (en) 2007-08-01 Retina-specific cells differentiated in vitro from bone marrow stem cells, the production thereof and their use
Hsueh et al. 2020 Lysophosphatidic acid improves corneal endothelial density in tissue culture by stimulating stromal secretion of interleukin‐1β
CN103571793A (en) 2014-02-12 Method of regulating and controlling retinal progenitor cell generated by inducing
Zeng et al. 2024 Distinctive small molecules blend: Promotes lacrimal gland epithelial cell proliferation in vitro and accelerates lacrimal gland injury repair in vivo
Dhamodaran et al. 2013 Ocular stem cells: an overview
WO2014179799A2 (en) 2014-11-06 Retina extracellular matrix based biomaterial
Lorenzo et al. 2013 Trypsin for dissociation of limbal cells for engineering of grafts may induce DNA strand breaks in the harvested cells
Riazuddin et al. 2022 The Function of Stem Cells in Ocular Homeostasis
Zhou et al. 2024 MicroRNA‐146a‐5p protects retinal ganglion cells through reducing neuroinflammation in experimental glaucoma
Zhang et al. 2020 Human Umbilical Cord Mesenchymal Stem Cell-derived Exosomal miR-27b Attenuate Subretinal Fibrosis via Suppressing Epithelial-mesenchymal Transition by Targeting HOXC6

Legal Events

Date Code Title Description
2019-03-19 PB01 Publication
2019-03-19 PB01 Publication
2019-04-12 SE01 Entry into force of request for substantive examination
2019-04-12 SE01 Entry into force of request for substantive examination
2021-05-07 GR01 Patent grant
2021-05-07 GR01 Patent grant
2024-10-18 OL01 Intention to license declared
2024-10-18 OL01 Intention to license declared