patents.google.com

CN108998411A - The isolated culture method of people's endometrial tissue derived mesenchymal stem cell - Google Patents

️Fri Dec 14 2018

The isolated culture method of people's endometrial tissue derived mesenchymal stem cell Download PDF

Info

Publication number

CN108998411A

CN108998411A CN201810930181.6A CN201810930181A CN108998411A CN 108998411 A CN108998411 A CN 108998411A CN 201810930181 A CN201810930181 A CN 201810930181A CN 108998411 A CN108998411 A CN 108998411A Authority

China

Prior art keywords

mesenchymal stem

cell

endometrial tissue

people

stem cell

Prior art date

2018-08-15

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.)

Pending

Application number

CN201810930181.6A

Other languages

Chinese (zh)

Inventor

解军

李仁科

范雪梅

宋慧芳

何生

平毅

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.)

Shanxi Medical University

Original Assignee

Shanxi Medical 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.)

2018-08-15

Filing date

2018-08-15

Publication date

2018-12-14

2018-08-15 Application filed by Shanxi Medical University filed Critical Shanxi Medical University

2018-08-15 Priority to CN201810930181.6A priority Critical patent/CN108998411A/en

2018-12-14 Publication of CN108998411A publication Critical patent/CN108998411A/en

Status Pending legal-status Critical Current

Links

210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 103
230000002357 endometrial effect Effects 0.000 title claims abstract description 93
238000012136 culture method Methods 0.000 title claims description 9
210000004027 cell Anatomy 0.000 claims abstract description 66
239000006285 cell suspension Substances 0.000 claims abstract description 25
208000015122 neurodegenerative disease Diseases 0.000 claims abstract description 9
238000011282 treatment Methods 0.000 claims abstract description 8
230000003716 rejuvenation Effects 0.000 claims abstract description 6
229940079593 drug Drugs 0.000 claims abstract description 5
239000003814 drug Substances 0.000 claims abstract description 5
208000023589 ischemic disease Diseases 0.000 claims abstract description 5
238000002360 preparation method Methods 0.000 claims abstract description 5
230000002265 prevention Effects 0.000 claims abstract description 5
238000000432 density-gradient centrifugation Methods 0.000 claims abstract description 3
239000007788 liquid Substances 0.000 claims description 28
230000029087 digestion Effects 0.000 claims description 24
239000006144 Dulbecco’s modiﬁed Eagle's medium Substances 0.000 claims description 23
210000000130 stem cell Anatomy 0.000 claims description 17
238000000034 method Methods 0.000 claims description 14
230000001079 digestive effect Effects 0.000 claims description 10
238000002955 isolation Methods 0.000 claims description 9
239000000047 product Substances 0.000 claims description 9
239000000872 buffer Substances 0.000 claims description 8
238000005119 centrifugation Methods 0.000 claims description 7
201000010099 disease Diseases 0.000 claims description 7
208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 7
239000006228 supernatant Substances 0.000 claims description 7
102000007260 Deoxyribonuclease I Human genes 0.000 claims description 6
108010008532 Deoxyribonuclease I Proteins 0.000 claims description 6
235000011389 fruit/vegetable juice Nutrition 0.000 claims description 6
239000007853 buffer solution Substances 0.000 claims description 4
230000002062 proliferating effect Effects 0.000 claims description 4
238000000926 separation method Methods 0.000 claims description 4
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 claims description 3
239000006143 cell culture medium Substances 0.000 claims description 3
239000001963 growth medium Substances 0.000 claims description 3
229960003531 phenolsulfonphthalein Drugs 0.000 claims description 3
230000009514 concussion Effects 0.000 claims 2
230000002255 enzymatic effect Effects 0.000 claims 2
238000003780 insertion Methods 0.000 claims 2
230000037431 insertion Effects 0.000 claims 2
210000003716 mesoderm Anatomy 0.000 claims 2
230000001419 dependent effect Effects 0.000 claims 1
230000035755 proliferation Effects 0.000 abstract description 13
230000004069 differentiation Effects 0.000 abstract description 11
230000005012 migration Effects 0.000 abstract description 7
238000013508 migration Methods 0.000 abstract description 7
208000028389 Nerve injury Diseases 0.000 abstract description 4
230000008764 nerve damage Effects 0.000 abstract description 4
230000004770 neurodegeneration Effects 0.000 abstract description 4
238000000746 purification Methods 0.000 abstract description 4
230000036541 health Effects 0.000 abstract description 3
230000006862 enzymatic digestion Effects 0.000 abstract 1
210000001519 tissue Anatomy 0.000 description 38
239000000243 solution Substances 0.000 description 22
239000002609 medium Substances 0.000 description 17
230000008439 repair process Effects 0.000 description 14
238000000338 in vitro Methods 0.000 description 10
230000008929 regeneration Effects 0.000 description 10
238000011069 regeneration method Methods 0.000 description 10
WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 8
210000001185 bone marrow Anatomy 0.000 description 8
238000012258 culturing Methods 0.000 description 8
230000009818 osteogenic differentiation Effects 0.000 description 8
238000002474 experimental method Methods 0.000 description 7
230000006698 induction Effects 0.000 description 7
101100298998 Caenorhabditis elegans pbs-3 gene Proteins 0.000 description 6
239000000203 mixture Substances 0.000 description 6
230000009815 adipogenic differentiation Effects 0.000 description 5
230000006870 function Effects 0.000 description 5
210000004291 uterus Anatomy 0.000 description 5
XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
102100022464 5'-nucleotidase Human genes 0.000 description 4
102100032912 CD44 antigen Human genes 0.000 description 4
102000029816 Collagenase Human genes 0.000 description 4
108060005980 Collagenase Proteins 0.000 description 4
102100037241 Endoglin Human genes 0.000 description 4
102000006354 HLA-DR Antigens Human genes 0.000 description 4
108010058597 HLA-DR Antigens Proteins 0.000 description 4
102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 4
101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 4
101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 4
101000881679 Homo sapiens Endoglin Proteins 0.000 description 4
101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 4
101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 4
101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 4
101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 4
102100022338 Integrin alpha-M Human genes 0.000 description 4
102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 4
102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 4
229960002424 collagenase Drugs 0.000 description 4
239000010410 layer Substances 0.000 description 4
238000006386 neutralization reaction Methods 0.000 description 4
230000008569 process Effects 0.000 description 4
102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 3
101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 3
210000004504 adult stem cell Anatomy 0.000 description 3
230000006378 damage Effects 0.000 description 3
238000001514 detection method Methods 0.000 description 3
210000004696 endometrium Anatomy 0.000 description 3
238000010348 incorporation Methods 0.000 description 3
239000003550 marker Substances 0.000 description 3
208000010125 myocardial infarction Diseases 0.000 description 3
239000008188 pellet Substances 0.000 description 3
238000012360 testing method Methods 0.000 description 3
APIXJSLKIYYUKG-UHFFFAOYSA-N 3 Isobutyl 1 methylxanthine Chemical compound O=C1N(C)C(=O)N(CC(C)C)C2=C1N=CN2 APIXJSLKIYYUKG-UHFFFAOYSA-N 0.000 description 2
241000283707 Capra Species 0.000 description 2
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 2
241000699670 Mus sp. Species 0.000 description 2
229930040373 Paraformaldehyde Natural products 0.000 description 2
102000004142 Trypsin Human genes 0.000 description 2
108090000631 Trypsin Proteins 0.000 description 2
230000002293 adipogenic effect Effects 0.000 description 2
239000008280 blood Substances 0.000 description 2
210000004369 blood Anatomy 0.000 description 2
230000007211 cardiovascular event Effects 0.000 description 2
230000012292 cell migration Effects 0.000 description 2
206010008118 cerebral infarction Diseases 0.000 description 2
208000026106 cerebrovascular disease Diseases 0.000 description 2
230000000694 effects Effects 0.000 description 2
238000000684 flow cytometry Methods 0.000 description 2
239000012634 fragment Substances 0.000 description 2
229940088597 hormone Drugs 0.000 description 2
239000005556 hormone Substances 0.000 description 2
239000012535 impurity Substances 0.000 description 2
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 description 2
208000037906 ischaemic injury Diseases 0.000 description 2
230000009245 menopause Effects 0.000 description 2
238000012986 modification Methods 0.000 description 2
230000004048 modification Effects 0.000 description 2
229920002866 paraformaldehyde Polymers 0.000 description 2
238000011160 research Methods 0.000 description 2
JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-UHFFFAOYSA-N 0.000 description 2
238000002054 transplantation Methods 0.000 description 2
239000012588 trypsin Substances 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 1
102000004877 Insulin Human genes 0.000 description 1
108090001061 Insulin Proteins 0.000 description 1
NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 0.000 description 1
230000001133 acceleration Effects 0.000 description 1
RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 1
230000033115 angiogenesis Effects 0.000 description 1
230000008901 benefit Effects 0.000 description 1
210000003855 cell nucleus Anatomy 0.000 description 1
230000008859 change Effects 0.000 description 1
UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
229960003957 dexamethasone Drugs 0.000 description 1
206010012601 diabetes mellitus Diseases 0.000 description 1
238000010586 diagram Methods 0.000 description 1
230000006806 disease prevention Effects 0.000 description 1
238000001035 drying Methods 0.000 description 1
210000001671 embryonic stem cell Anatomy 0.000 description 1
230000004217 heart function Effects 0.000 description 1
230000005847 immunogenicity Effects 0.000 description 1
230000006872 improvement Effects 0.000 description 1
229940125396 insulin Drugs 0.000 description 1
208000028867 ischemia Diseases 0.000 description 1
230000000302 ischemic effect Effects 0.000 description 1
239000002346 layers by function Substances 0.000 description 1
239000012528 membrane Substances 0.000 description 1
230000003821 menstrual periods Effects 0.000 description 1
230000000877 morphologic effect Effects 0.000 description 1
239000012120 mounting media Substances 0.000 description 1
230000000737 periodic effect Effects 0.000 description 1
238000004393 prognosis Methods 0.000 description 1
230000001737 promoting effect Effects 0.000 description 1
238000010791 quenching Methods 0.000 description 1
230000000171 quenching effect Effects 0.000 description 1
230000009257 reactivity Effects 0.000 description 1
230000001172 regenerating effect Effects 0.000 description 1
230000007651 self-proliferation Effects 0.000 description 1
210000002966 serum Anatomy 0.000 description 1
239000012679 serum free medium Substances 0.000 description 1
238000010186 staining Methods 0.000 description 1
238000011476 stem cell transplantation Methods 0.000 description 1
230000009469 supplementation Effects 0.000 description 1
230000001225 therapeutic effect Effects 0.000 description 1
238000002560 therapeutic procedure Methods 0.000 description 1
238000012546 transfer Methods 0.000 description 1
GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
239000012224 working solution Substances 0.000 description 1

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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- 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
- C12N2500/02—Atmosphere, e.g. low oxygen conditions
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Landscapes

Health & Medical Sciences (AREA)
Life Sciences & Earth Sciences (AREA)
Engineering & Computer Science (AREA)
Bioinformatics & Cheminformatics (AREA)
Chemical & Material Sciences (AREA)
Biomedical Technology (AREA)
Organic Chemistry (AREA)
General Health & Medical Sciences (AREA)
Veterinary Medicine (AREA)
Medicinal Chemistry (AREA)
Pharmacology & Pharmacy (AREA)
Animal Behavior & Ethology (AREA)
Public Health (AREA)
Developmental Biology & Embryology (AREA)
Zoology (AREA)
Chemical Kinetics & Catalysis (AREA)
General Chemical & Material Sciences (AREA)
Cell Biology (AREA)
Biotechnology (AREA)
Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
Neurosurgery (AREA)
Wood Science & Technology (AREA)
Neurology (AREA)
Immunology (AREA)
Genetics & Genomics (AREA)
Virology (AREA)
Epidemiology (AREA)
Hospice & Palliative Care (AREA)
Hematology (AREA)
General Engineering & Computer Science (AREA)
Psychiatry (AREA)
Biochemistry (AREA)
Microbiology (AREA)
Rheumatology (AREA)
Urology & Nephrology (AREA)
Vascular Medicine (AREA)
Cardiology (AREA)
Heart & Thoracic Surgery (AREA)
Dermatology (AREA)
Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

本发明涉及一种人子宫内膜组织来源间充质干细胞，该子宫内膜组织来源的间充质干细胞是从人的子宫内膜组织中分离得到。通过酶解法消化人体子宫内膜组织获得单细胞悬液后，直接接种于间充质干细胞培养基中进行培养及传代纯化，或者采用密度梯度离心法分离得到包含有间充质干细胞的细胞群，接种于间充质干细胞培养基中，进行培养及传代纯化，最终获得了新的具有强增殖能力及迁移分化能力的间充质干细胞。本发明的间充质干细胞可以作为制备预防或治疗缺血性疾病、神经损伤、神经退行性疾病、其他年龄相关退行性疾病的药物得到应用，也可以用于制备包括医学美容、亚健康人群保健、老年个体年轻化等方面的产品。

The invention relates to a human endometrial tissue-derived mesenchymal stem cell, which is isolated from human endometrial tissue. After enzymatic digestion of human endometrial tissue to obtain a single cell suspension, it is directly inoculated in a mesenchymal stem cell medium for culture and passage purification, or the cell population containing mesenchymal stem cells is obtained by density gradient centrifugation. Inoculated in mesenchymal stem cell medium, cultured and subcultured, and finally obtained new mesenchymal stem cells with strong proliferation ability, migration and differentiation ability. The mesenchymal stem cells of the present invention can be used as medicines for the prevention or treatment of ischemic diseases, nerve damage, neurodegenerative diseases, and other age-related degenerative diseases, and can also be used for the preparation of medicines including medical cosmetology and health care for sub-healthy people. , Rejuvenation of elderly individuals and other products.

Description

人子宫内膜组织来源间充质干细胞的分离培养方法Isolation and culture method of mesenchymal stem cells derived from human endometrial tissue

技术领域technical field

本发明涉及一种新的干细胞，特别是一种从人体子宫内膜组织中提取的间充质干细胞。本发明还涉及该子宫内膜间充质干细胞的分离培养方法。The invention relates to a new stem cell, in particular to a mesenchymal stem cell extracted from human endometrial tissue. The invention also relates to a method for isolating and culturing the endometrial mesenchymal stem cells.

背景技术Background technique

干细胞是一种具有自我复制、更新和多向分化潜能的细胞，具有修复受损组织细胞的潜能，近年来已成为疾病治疗和组织工程领域理想的种子细胞，是各种疾病尤其是各种难治性疾病可能的新的有效治疗方法。Stem cells are a kind of cells with self-replication, renewal and multi-directional differentiation potential, and have the potential to repair damaged tissue cells. In recent years, they have become ideal seed cells in the field of disease treatment and tissue engineering. Potentially new and effective treatments for diseases.

根据发育阶段不同，干细胞又分为胚胎干细胞和成体干细胞，其中成体干细胞因其来源广泛，致瘤性相对较低等特点，具有广泛的应用前景。According to different developmental stages, stem cells are divided into embryonic stem cells and adult stem cells. Adult stem cells have broad application prospects due to their wide range of sources and relatively low tumorigenicity.

成体干细胞中的间充质干细胞更因其易分离培养，扩增纯化，免疫原性低等特点，成为干细胞移植及组织工程领域理想的种子细胞。不仅为包括心梗、脑梗等在内的缺血性疾病、神经损伤、神经退行性疾病、其他年龄相关的退行性疾病及其他各种不同疾病的治疗提供了新的希望，同时在医学美容领域，亚健康人群自我保健，乃至老年个体年轻化等方面都提供了新的思路。Among adult stem cells, mesenchymal stem cells are ideal seed cells for stem cell transplantation and tissue engineering because of their easy isolation and culture, expansion and purification, and low immunogenicity. It not only provides new hope for the treatment of ischemic diseases including myocardial infarction, cerebral infarction, nerve injury, neurodegenerative disease, other age-related degenerative diseases and other various diseases, but also in medical aesthetics It provides new ideas in areas such as self-care for sub-healthy people, and even the rejuvenation of elderly individuals.

研究表明，间充质干细胞具有促进损伤修复、改善多种疾病预后的功能。增殖活力旺盛的年轻间充质干细胞进行移植，能够促使受体对修复干细胞的反应性增强，促进受体的年轻化。间充质干细胞发挥其修复治疗功能需要具备两个要素，一是数量充足，一是包括自我更新增殖等能力在内的功能旺盛活跃。Studies have shown that mesenchymal stem cells can promote damage repair and improve the prognosis of various diseases. The transplantation of young mesenchymal stem cells with vigorous proliferative activity can enhance the reactivity of recipients to repair stem cells and promote the rejuvenation of recipients. Mesenchymal stem cells need to have two elements to exert their repairing and therapeutic functions, one is sufficient quantity, and the other is vigorous and active functions including the ability of self-renewal and proliferation.

目前已有的间充质干细胞主要包括骨髓间充质干细胞、脂肪间充质干细胞等，研究结果显示它们具有较好的促进损伤修复及疾病预防、治疗前景。At present, the existing mesenchymal stem cells mainly include bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, etc. The research results show that they have good prospects for promoting damage repair, disease prevention and treatment.

发明内容Contents of the invention

本发明的目的是提供一种新的具有强修复再生能力的人子宫内膜组织来源间充质干细胞。The purpose of the present invention is to provide a new human endometrial tissue-derived mesenchymal stem cell with strong repair and regeneration ability.

提供所述人子宫内膜组织来源间充质干细胞的分离培养方法，是本发明的另一发明目的。Another object of the present invention is to provide a method for isolating and culturing the human endometrial tissue-derived mesenchymal stem cells.

本发明的目的还在于提供所述新的间充质干细胞的应用。The purpose of the present invention is also to provide the application of the new mesenchymal stem cells.

一种有趣的临床现象给发明人提供了新的重要提示：绝经前女性的心血管事件发生率明显低于同年龄段的男性，但女性这种发病率低的优势会在绝经后丧失。An interesting clinical phenomenon provided the inventor with a new important hint: the incidence of cardiovascular events in premenopausal women is significantly lower than that of men of the same age, but the advantage of low incidence in women will be lost after menopause.

现有研究结果显示，虽然绝经前后女性激素水平发生巨大变化，但单纯补充激素治疗并不能明显遏制绝经后女性心血管事件发生率增加的态势。另一方面，子宫内膜具有超强的再生能力，其功能层会发生周期性增殖、分泌和脱落，即绝经前女性所具有的生理期周期性变化。以上现象均提示，人体子宫中具有再生能力很强的干细胞。Existing research results show that although hormone levels in women undergo tremendous changes before and after menopause, hormone supplementation alone cannot significantly curb the increase in the incidence of cardiovascular events in postmenopausal women. On the other hand, the endometrium has a strong regenerative ability, and its functional layer will proliferate, secrete and shed periodically, which is the periodic change of menstrual period in premenopausal women. The above phenomena all suggest that there are stem cells with strong regeneration ability in the human uterus.

在绝经前女性、绝经后女性以及男性三种人群中均具有骨髓及脂肪等其他来源的间充质干细胞，而导致上述这种发病率差异的原因，很可能是三种人群中是否具有子宫中再生能力很强的干细胞，同时预示子宫中的这种干细胞具有很强的损伤修复功能。Premenopausal women, postmenopausal women, and men all have mesenchymal stem cells from other sources such as bone marrow and fat. Stem cells with strong regeneration ability also indicate that such stem cells in the uterus have a strong damage repair function.

发明人在前期的小鼠研究中已经观察到，小鼠心梗后，有子宫来源的细胞归巢到心脏缺血损伤局部，参与血管新生及缺血损伤修复，并进而改善缺血心脏的心功能。因此，在人体子宫中是否也存在具有强的修复再生能力的干细胞，以及如何分离、培养出人体子宫中这种强的修复再生能力的干细胞，并将其用于预防和临床治疗领域，是一项非常具有临床应用前景及挑战性的工作。The inventors have observed in previous studies on mice that after myocardial infarction in mice, cells from the uterus would homing to the ischemic injury area of the heart, participate in angiogenesis and repair of ischemic injury, and then improve the cardiac function of the ischemic heart. Function. Therefore, whether there are stem cells with strong repair and regeneration ability in the human uterus, and how to isolate and cultivate such stem cells with strong repair and regeneration ability in the human uterus, and use them in the field of prevention and clinical treatment, is an important issue. This is a very promising and challenging work for clinical application.

首先，本发明提出了一种新的具有强修复再生能力的人子宫内膜组织来源间充质干细胞，该子宫内膜组织来源的间充质干细胞能够从人的子宫内膜组织中分离得到。Firstly, the present invention proposes a novel human endometrial tissue-derived mesenchymal stem cell with strong repair and regeneration ability, and the endometrial tissue-derived mesenchymal stem cell can be isolated from human endometrial tissue.

其次，本发明提供了一种所述新的具有强修复再生能力的人子宫内膜组织来源间充质干细胞的分离培养方法，通过酶解法消化人体子宫内膜组织获得单细胞悬液后，直接接种于间充质干细胞培养基中进行培养及传代纯化，最终获得了上述新的具有强修复再生能力的间充质干细胞。Secondly, the present invention provides a method for isolating and culturing the novel human endometrial tissue-derived mesenchymal stem cells with strong repair and regeneration ability. Inoculated in the medium of mesenchymal stem cells for culture and passage purification, and finally obtained the above-mentioned new mesenchymal stem cells with strong repair and regeneration ability.

具体地，本发明所述人子宫内膜组织来源间充质干细胞的分离培养方法是按照下述步骤进行的。Specifically, the method for isolating and culturing human endometrial tissue-derived mesenchymal stem cells according to the present invention is carried out according to the following steps.

1）以含250～350µg/ml胶原酶Ⅲ和30～50µg/ml DNase I的PBS缓冲液为消化液，将所述消化液与人子宫内膜组织按照2～4∶1的体积比混合，37℃水浴震荡消化40～60min后，抽取消化产物上清液，以1∶1的体积比加入10vol% FBS-DMEM/F12进行中和，终止消化后获得单细胞悬液。1) Use PBS buffer containing 250-350 µg/ml collagenase III and 30-50 µg/ml DNase I as the digestive solution, mix the digestive solution with human endometrial tissue at a volume ratio of 2-4:1, After digesting with shaking in a water bath at 37°C for 40-60 min, extract the supernatant of the digested product, add 10vol% FBS-DMEM/F12 at a volume ratio of 1:1 for neutralization, and obtain a single-cell suspension after terminating the digestion.

2）将所述单细胞悬液以200目细胞滤网过滤，1000～1200r/min离心获得细胞团块。2) Filter the single cell suspension with a 200-mesh cell strainer, and centrifuge at 1000-1200 r/min to obtain cell aggregates.

3）在所述细胞团块中加入10vol% FBS-DMEM/F12培养基，37℃、5%CO₂细胞培养箱中培养，经不少于1次的传代培养纯化，分离得到人子宫内膜组织来源间充质干细胞。3) Add 10vol% FBS-DMEM/F12 medium to the cell mass, culture in a 37°C, 5% CO ₂ cell incubator, and purify through no less than one subculture to obtain human endometrium Tissue-derived mesenchymal stem cells.

进而，本发明还提供了另外一种所述新的具有强修复再生能力的人子宫内膜组织来源间充质干细胞的分离培养方法，通过酶解法消化人体子宫内膜组织获得单细胞悬液后，采用密度梯度离心法分离得到包含有间充质干细胞在内的细胞群，接种于间充质干细胞培养基中，经培养及传代纯化，最终获得了上述新的具有强修复再生能力的间充质干细胞。Furthermore, the present invention also provides another method for isolating and culturing human endometrial tissue-derived mesenchymal stem cells with strong repair and regeneration capabilities. After enzymatically digesting human endometrial tissue to obtain a single cell suspension , the cell population containing mesenchymal stem cells was separated by density gradient centrifugation, inoculated in mesenchymal stem cell culture medium, cultivated and subcultured, and finally obtained the above-mentioned new mesenchymal stem cells with strong repair and regeneration ability. stem cells.

3）分别配制密度为1.048～1.052g/ml，1.058～1.062g/ml，1.070～1.084g/ml的三种含有Percoll和Ads的梯度密度缓冲液，分别记为A液、B液和C液，其中B液以0.01g/l酚红标识。3) Prepare three gradient density buffer solutions containing Percoll and Ads with densities of 1.048-1.052g/ml, 1.058-1.062g/ml, and 1.070-1.084g/ml respectively, and record them as solution A, solution B and solution C respectively , where liquid B is marked with 0.01g/l phenol red.

4）在加有A液的离心管中移入等体积的B液，轻轻插入离心管底部缓慢滴加在A液下方，形成两层液体分离的界面。4) Transfer an equal volume of liquid B into the centrifuge tube added with liquid A, gently insert the bottom of the centrifuge tube and slowly drop it under liquid A to form an interface where two layers of liquid separate.

5）用与A液等体积的C液重悬步骤2）获得的细胞团块，得到细胞悬液，将所述细胞悬液插入离心管底部缓慢加入在B液下方，形成具有无色-红色-无色三层液体清晰分离界面的梯度密度缓冲液。5) Resuspend the cell mass obtained in step 2) with liquid C equal to the volume of liquid A to obtain a cell suspension, insert the cell suspension into the bottom of a centrifuge tube and slowly add it under liquid B to form a colorless-red - Gradient density buffer with clear separation interface of colorless three-layer liquid.

6）以1300～1700r/min离心，将细胞分散在梯度密度缓冲液的不同密度区域，吸取B液下方和C液上方区域的细胞悬液，放入预先加入15～30ml 10vol% FBS-DMEM/F12的离心管中，轻柔混匀。6) Centrifuge at 1300-1700r/min, disperse the cells in different density areas of the gradient density buffer, absorb the cell suspension in the area below the B solution and above the C solution, and put it into the pre-added 15-30ml 10vol% FBS-DMEM/ In a centrifuge tube of F12, mix gently.

7）1000～1300r/min离心获得细胞团块，加入10～20ml 10vol% FBS-DMEM/F12，轻柔混匀，1000～1300r/min离心并收集细胞团块。7) Centrifuge at 1000-1300r/min to obtain cell agglomerates, add 10-20ml 10vol% FBS-DMEM/F12, mix gently, centrifuge at 1000-1300r/min and collect cell agglomerates.

8）以10vol% FBS-DMEM/F12培养基重悬细胞团块，接种于培养瓶或培养皿中，37℃、5%CO₂细胞培养箱中培养，经不少于1次的传代培养纯化，分离得到人子宫内膜组织来源间充质干细胞。8) Resuspend the cell mass in 10vol% FBS-DMEM/F12 medium, inoculate it in a culture bottle or a culture dish, culture it in a cell incubator at 37°C and 5% CO ₂ , and purify it through no less than one subculture , isolated human endometrial tissue-derived mesenchymal stem cells.

本发明上述任意一种人子宫内膜组织来源间充质干细胞的分离培养过程中，除指定温度外，其余操作均在室温下进行。During the isolation and culture process of any one of the above-mentioned human endometrial tissue-derived mesenchymal stem cells in the present invention, except for the specified temperature, all other operations are carried out at room temperature.

本发明上述任意一种人子宫内膜组织来源间充质干细胞的分离培养方法中，所使用的人子宫内膜组织是以PBS清洗干净，并充分切碎的组织。In any one of the above methods for isolating and culturing mesenchymal stem cells derived from human endometrial tissue of the present invention, the human endometrial tissue used is cleaned with PBS and fully chopped.

其中，任意一种人子宫内膜组织来源间充质干细胞的分离培养方法中，是将所述人子宫内膜组织以所述消化液消化不少于2次，并合并消化得到的细胞悬液。Wherein, in any one of the methods for isolating and culturing human endometrial tissue-derived mesenchymal stem cells, the human endometrial tissue is digested no less than twice with the digestive juice, and the cell suspension obtained from the digestion is combined .

本发明分离、培养、纯化获取的人子宫内膜间充质干细胞具有很强的增殖能力及迁移分化能力，可以作为制备预防或治疗包括心梗、脑梗、糖尿病肢体远端缺血等在内的缺血性疾病、神经损伤、神经退行性疾病、其他年龄相关的退行性疾病的药物得到应用，也可以用于制备包括医学美容、亚健康人群保健、老年个体年轻化等方面的产品。The human endometrial mesenchymal stem cells isolated, cultivated and purified by the present invention have strong proliferation ability and migration and differentiation ability, and can be used as a preparation for prevention or treatment including myocardial infarction, cerebral infarction, and distal ischemia in diabetic limbs. Drugs for ischemic diseases, nerve damage, neurodegenerative diseases, and other age-related degenerative diseases can also be used to prepare products including medical cosmetology, health care for sub-healthy people, and rejuvenation of elderly individuals.

体外细胞实验证明，通过上述体外分离培养方法获得的人子宫内膜来源的间充质干细胞可以在体外传代、扩增，具有干细胞自我增殖更新的基本特性。In vitro cell experiments have proved that the human endometrium-derived mesenchymal stem cells obtained by the above in vitro isolation and culture method can be passaged and expanded in vitro, and have the basic characteristics of self-proliferation and renewal of stem cells.

进而，选择间充质干细胞表面标志物CD90、CD44、CD73、CD105及PE-negative（包括CD34 PE、CD11b PE、CD19 PE、CD45 PE和HLA-DR PE），对上述体外分离培养的人子宫内膜间充质干细胞进行流式细胞术检测，鉴定结果显示CD90⁺，CD44⁺，CD73⁺，CD105⁺以及CD34^- ,CD11b^- ,CD19^- , CD45^-，HLA-DR^-，相应标志物的阳性率和阴性率均符合间充质干细胞的表面标志物特点。Furthermore, the surface markers of mesenchymal stem cells CD90, CD44, CD73, CD105 and PE-negative (including CD34 PE, CD11b PE, CD19 PE, CD45 PE and HLA-DR PE) were selected, and the human intrauterine Membrane mesenchymal stem cells were detected by flow cytometry, and the identification results showed the positive rates of CD90 ⁺ , CD44 ⁺ , CD73 ⁺ , CD105 ⁺ and CD34 ^- , CD11b ^- , CD19 ^- , CD45 ^- , HLA-DR ^- , and corresponding markers Both the negative rate and the negative rate were consistent with the surface marker characteristics of mesenchymal stem cells.

通过BrdU掺入实验、划痕实验及体外诱导分化实验，均证实了本发明获取的人子宫内膜间充质干细胞具有增殖、迁移、诱导分化等功能。Through BrdU incorporation experiments, scratch experiments, and in vitro differentiation induction experiments, it has been confirmed that the human endometrial mesenchymal stem cells obtained by the present invention have functions such as proliferation, migration, and induction of differentiation.

更为重要的是，BrdU掺入实验结果显示，与同年龄段、相同代数的骨髓间充质干细胞相比，本发明人子宫内膜间充质干细胞具有更强的增殖能力，即具有更强的活力，具备快速获得更多种子细胞的能力。More importantly, the results of BrdU incorporation experiments show that compared with bone marrow mesenchymal stem cells of the same age group and the same generation, the human endometrial mesenchymal stem cells of the present invention have stronger proliferation ability, that is, they have stronger Vitality, with the ability to quickly obtain more seed cells.

附图说明Description of drawings

图1是体外培养人子宫内膜间充质干细胞细胞形态图。Figure 1 is a morphological diagram of human endometrial mesenchymal stem cells cultured in vitro.

图2是富集的人子宫内膜间充质干细胞的流式鉴定结果柱状图。Fig. 2 is a histogram of flow cytometric identification results of enriched human endometrial mesenchymal stem cells.

图3是人子宫内膜间充质干细胞划痕实验显示迁移能力的结果图。Fig. 3 is a graph showing the migration ability of human endometrial mesenchymal stem cells by a scratch test.

图4是人子宫内膜间充质干细胞成骨诱导分化的结果图。Fig. 4 is a graph showing the results of osteogenic differentiation of human endometrial mesenchymal stem cells.

图5是人子宫内膜间充质干细胞成脂诱导分化的结果图。Fig. 5 is a graph showing the results of adipogenic induction and differentiation of human endometrial mesenchymal stem cells.

图6是人子宫内膜间充质干细胞BrdU掺入实验显示增殖能力的结果图。Fig. 6 is a graph showing the results of proliferation ability of human endometrial mesenchymal stem cells by BrdU incorporation experiment.

具体实施方式Detailed ways

下述实施例仅为本发明的优选技术方案，并不用于对本发明进行任何限制。对于本领域技术人员而言，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The following examples are only preferred technical solutions of the present invention, and are not intended to limit the present invention in any way. Various modifications and variations of the present invention will occur to those skilled in the art. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

实施例1：人子宫内膜间充质干细胞的分离培养。Example 1: Isolation and culture of human endometrial mesenchymal stem cells.

1）获取人子宫内膜组织，放置于无菌的1×PBS中，尽快移入超净操作台。1) Obtain human endometrial tissue, place it in sterile 1×PBS, and move it into the ultra-clean operating table as soon as possible.

2）无菌培养器皿中，将人子宫内膜组织用PBS反复清洗3次，去除掉表面血液等杂物。2) In a sterile culture vessel, the human endometrial tissue was repeatedly washed with PBS 3 times to remove surface blood and other impurities.

3）将清洗干净的人子宫内膜组织充分切碎至小于1mm³。3) Fully chop the cleaned human endometrial tissue to less than 1mm ³ .

4）向切碎的人子宫内膜组织中加入3倍体积含300µg/ml胶原酶Ⅲ和40µg/mlDNase I的PBS消化液，以110rpm的震荡速度在37℃水浴中震荡消化45min，抽取消化产物上清液，以1∶1的体积比加入10% FBS-DMEM/F12进行中和以终止消化。4) Add 3 times the volume of PBS digestion solution containing 300 µg/ml collagenase III and 40 µg/ml DNase I to the minced human endometrial tissue, shake and digest in a 37°C water bath at a shaking speed of 110rpm for 45min, and extract the digested product The supernatant was neutralized by adding 10% FBS-DMEM/F12 at a volume ratio of 1:1 to terminate the digestion.

5）在未消化的组织碎片中再次加入消化液，重复步骤4）的消化过程。5) Add digestion solution to the undigested tissue fragments again, and repeat the digestion process of step 4).

6）将上述两步消化及中和后得到的单细胞悬液以200目细胞滤网过滤，1000r/min离心5min，获得细胞团块。6) Filter the single cell suspension obtained after the above two steps of digestion and neutralization with a 200-mesh cell strainer, and centrifuge at 1000r/min for 5min to obtain cell pellets.

7）以10% FBS-DMEM/F12培养基重悬分离获得的细胞团块，计数，37℃、5%CO₂细胞培养箱中培养。7) Resuspend the isolated cell aggregates in 10% FBS-DMEM/F12 medium, count them, and culture them in a cell incubator at 37°C and 5% CO ₂ .

8）待细胞生长至90%融合时，以含0.25%胰酶和0.02%EDTA的PBS消化液消化，传代，继续以10% FBS-DMEM/F12培养基培养。至第3代，得到纯化的人子宫内膜组织来源间充质干细胞。8) When the cells grow to 90% confluence, they are digested with PBS digestion solution containing 0.25% trypsin and 0.02% EDTA, passaged, and continue to be cultured in 10% FBS-DMEM/F12 medium. Up to the third passage, purified human endometrial tissue-derived mesenchymal stem cells were obtained.

实施例2：人子宫内膜间充质干细胞的分离培养。Example 2: Isolation and culture of human endometrial mesenchymal stem cells.

3）将清洗干净的人子宫内膜组织充分切碎至小于1mm³。3) Fully chop the cleaned human endometrial tissue to less than 1mm ³ .

7）配制3种含有Percoll和Ads的梯度密度缓冲液（A液\B液\C液），各缓冲液密度分别为1.050g/ml，1.060g/ml，1.082g/ml，其中B液以0.01g/l酚红标识为红色。7) Prepare 3 kinds of gradient density buffer solutions containing Percoll and Ads (A solution\B solution\C solution). 0.01g/l phenol red is marked as red.

8）取10ml A液，加入新的50ml离心管的底部。8) Take 10ml of solution A and add it to the bottom of a new 50ml centrifuge tube.

9）将吸有10ml B液的移液管轻轻插入离心管底部，轻柔缓慢滴加，形成两层液体分离的界面。9) Gently insert the pipette with 10ml of liquid B into the bottom of the centrifuge tube, and add gently and slowly to form an interface where two layers of liquid separate.

10）用10ml C液重悬获得的细胞团块得到细胞悬液，吸取细胞悬液，插入离心管底部缓慢加入在B液下方，形成具有无色-红色-无色三层液体清晰分离界面的梯度密度缓冲液。10) Resuspend the obtained cell aggregates with 10ml of liquid C to obtain a cell suspension, absorb the cell suspension, insert it into the bottom of the centrifuge tube and slowly add it under the liquid B to form a clear separation interface of colorless-red-colorless three-layer liquid Gradient density buffer.

11）以转速1500r/min，保证加速度减速度为0，室温离心35min。11) Centrifuge at room temperature for 35 minutes at a rotational speed of 1500r/min to ensure that the acceleration and deceleration are 0.

12）离心完毕后，吸取位于B液下方和C液上方区域的细胞悬液，放入预先加入20ml10% FBS-DMEM-F12的50ml离心管中，轻柔混匀。12) After centrifugation, pipette the cell suspension located below the solution B and above the solution C, put it into a 50ml centrifuge tube with 20ml 10% FBS-DMEM-F12 added in advance, and mix gently.

13）继续在转速1200r/min下，室温离心10min。13) Continue to centrifuge at room temperature for 10 minutes at a rotational speed of 1200r/min.

14）弃去上清，再加入10ml 10% FBS-DMEM/F12，轻柔混匀，1200r/min下，室温离心5min得到细胞团块。14) Discard the supernatant, then add 10ml 10% FBS-DMEM/F12, mix gently, and centrifuge at 1200r/min for 5min at room temperature to obtain cell pellets.

15）以10% FBS-DMEM/F12培养基重悬分离获得的细胞团块，计数，37℃、5%CO₂细胞培养箱中培养。15) Resuspend the isolated cell aggregates in 10% FBS-DMEM/F12 medium, count them, and culture them in a cell incubator at 37°C and 5% CO ₂ .

16）细胞生长至90%融合时，以含0.25%胰酶和0.02%EDTA的PBS消化液消化，传代，继续以10% FBS-DMEM/F12培养基培养。至第3代，得到纯化的人子宫内膜组织来源间充质干细胞。16) When the cells grow to 90% confluence, they are digested with PBS digestion solution containing 0.25% trypsin and 0.02% EDTA, passaged, and continue to be cultured in 10% FBS-DMEM/F12 medium. Up to the third passage, purified human endometrial tissue-derived mesenchymal stem cells were obtained.

经过上述分离、培养、纯化后的人子宫内膜间充质干细胞，在正常生长状态下，倒置相差显微镜摄片，活细胞培养形态见图1，人子宫内膜间充质干细胞形态趋于一致，呈长梭形，漩涡状生长，具有间充质干细胞的特征性形态。The human endometrial mesenchymal stem cells after the above separation, culture, and purification were taken under a normal growth state by an inverted phase-contrast microscope. The cultured morphology of living cells is shown in Figure 1. , showing long fusiform, spiral growth, with the characteristic morphology of mesenchymal stem cells.

实施例3：人子宫内膜间充质干细胞的流式鉴定。Example 3: Flow cytometric identification of human endometrial mesenchymal stem cells.

1）人子宫内膜间充质干细胞表面标志物选择CD90，CD44，CD73，CD105以及PE-negative（包括CD34 PE，CD11b PE，CD19PE，CD45 PE和HLA-DR PE）。1) The surface markers of human endometrial mesenchymal stem cells are CD90, CD44, CD73, CD105 and PE-negative (including CD34 PE, CD11b PE, CD19PE, CD45 PE and HLA-DR PE).

2）取培养至第3代的人子宫内膜间充质干细胞，常规消化后，用上述表面标志物对应抗体染色。2) Human endometrial mesenchymal stem cells cultured to the third passage were taken, after routine digestion, and stained with antibodies corresponding to the above surface markers.

3）流式细胞术检测人子宫内膜间充质干细胞表面标志物。3) Detection of surface markers of human endometrial mesenchymal stem cells by flow cytometry.

图2给出了人子宫内膜间充质干细胞的流式结果鉴定，柱状图显示为每个表面标志物的阳性百分率。Figure 2 shows the flow cytometric identification of human endometrial mesenchymal stem cells, and the histogram shows the positive percentage of each surface marker.

图2显示，分离培养的人子宫内膜间充质干细胞呈现CD90⁺，CD44⁺，CD73⁺，CD105⁺以及CD34^-，CD11b^-，CD19^-，CD45^-，HLA-DR^-的表面标记物特征，且相应标志物的阳性率和阴性率均符合间充质干细胞的表面标志物特点，即从子宫内膜组织中分离、培养、纯化获得的干细胞是间充质干细胞。Figure 2 shows that the isolated and cultured human endometrial mesenchymal stem cells present CD90 ⁺ , CD44 ⁺ , CD73 ⁺ , CD105 ⁺ and CD34 ^- , CD11b ^- , CD19 ^- , CD45 ^- , HLA-DR ^- surface marker characteristics, And the positive rate and negative rate of the corresponding markers are in line with the characteristics of the surface markers of mesenchymal stem cells, that is, the stem cells isolated, cultured and purified from endometrial tissue are mesenchymal stem cells.

实施例4：人子宫内膜间充质干细胞的迁移能力鉴定。Example 4: Identification of migration ability of human endometrial mesenchymal stem cells.

1）人子宫内膜间充质干细胞接种于6孔板中，10% FBS-DMEM/F12培养基中培养至100%融合。1) Human endometrial mesenchymal stem cells were seeded in 6-well plates and cultured in 10% FBS-DMEM/F12 medium until 100% confluent.

2）无菌枪尖在孔内中央区划一纵向垂线。2) The sterile gun tip draws a vertical vertical line in the center of the hole.

3）PBS洗细胞3次，去除划下的细胞碎片。3) Wash the cells 3 times with PBS to remove the scratched cell debris.

4）无血清培养基培养24h，镜下观察，拍照。4) Cultivate in serum-free medium for 24 hours, observe under a microscope, and take pictures.

5）以Image J软件测量不同时间点的划痕宽度，计算细胞迁移率。5) Measure the scratch width at different time points with Image J software, and calculate the cell migration rate.

图3显示出分离培养获得的人子宫内膜间充质干细胞具有很好的迁移能力，24h迁移率达到77.52%±7.60%。Figure 3 shows that the isolated and cultured human endometrial mesenchymal stem cells have good migration ability, and the 24h migration rate reaches 77.52%±7.60%.

实施例5：人子宫内膜间充质干细胞成骨诱导分化能力鉴定。Example 5: Identification of osteogenic differentiation ability of human endometrial mesenchymal stem cells.

1）将人子宫内膜间充质干细胞以7×10³个细胞/cm²的密度接种到12孔板中，每孔加入1ml 10% FBS-DMEM/F12培养基，置于37℃、5%CO₂培养箱中正常培养。1) Seed human endometrial mesenchymal stem cells in a 12-well plate at a density of 7×10 ³ cells/cm ² , add 1 ml of 10% FBS-DMEM/F12 medium to each well, and place at 37°C for 5 normal culture in a %CO ₂ incubator.

2）24h后，去除培养基，加入BI快速成骨分化培养基1ml，继续置于37℃、5%CO₂培养箱内培养。2) After 24 hours, remove the medium, add 1ml of BI rapid osteogenic differentiation medium, and continue to culture in a 37°C, 5% CO ₂ incubator.

3）每3天更换一次成骨分化培养基，诱导成骨分化2周。3) The osteogenic differentiation medium was replaced every 3 days, and osteogenic differentiation was induced for 2 weeks.

4）诱导分化结束后，行茜素红染色，倒置显微镜下观察并拍照，分析成骨诱导分化能力。4) After induction and differentiation, perform alizarin red staining, observe and take pictures under an inverted microscope, and analyze the osteogenic differentiation ability.

图4显示人子宫内膜间充质干细胞在体外能够完成成骨诱导分化，具有成骨分化潜能。Figure 4 shows that human endometrial mesenchymal stem cells can complete osteogenic differentiation in vitro and have osteogenic differentiation potential.

实施例6：人子宫内膜间充质干细胞成脂诱导分化能力鉴定。Example 6: Identification of adipogenic differentiation ability of human endometrial mesenchymal stem cells.

2）待细胞达到100%融合后，去除培养基，加入1ml成脂诱导分化培养基(10%FBS，1µM地塞米松，10µg/ml胰岛素，0.5mM 1-methyl-3-isobutylxanthine(IBMX)和1µM罗格列)。2) After the cells reached 100% confluency, remove the medium and add 1ml of adipogenic differentiation medium (10% FBS, 1µM dexamethasone, 10µg/ml insulin, 0.5mM 1-methyl-3-isobutylxanthine (IBMX) and 1 µM Rogley).

3）每3天更换1次成脂诱导分化培养基，如此反复更换8次。3) The adipogenic differentiation medium was replaced every 3 days, and so repeated 8 times.

4）诱导分化结束后，行油红O染色，倒置显微镜下观察并拍照，分析成脂诱导分化能力。4) After induction of differentiation, stain with Oil Red O, observe and take pictures under an inverted microscope, and analyze the ability of adipogenic induction and differentiation.

图5显示出人子宫内膜间充质干细胞在体外能够完成成脂诱导分化，具有成脂分化潜能。图4与图5结合提示人子宫内膜间充质干细胞具有多向分化潜能。Figure 5 shows that human endometrial mesenchymal stem cells can complete adipogenic differentiation in vitro and have adipogenic differentiation potential. The combination of Figure 4 and Figure 5 suggests that human endometrial mesenchymal stem cells have multilineage differentiation potential.

实施例7：人子宫内膜间充质干细胞增殖能力鉴定。Example 7: Identification of proliferation ability of human endometrial mesenchymal stem cells.

1）将人子宫内膜间充质干细胞以7000个/孔的密度接种于加有无菌小圆玻片的24孔板中，细胞贴壁后，用含有10µM Brdu的10% FBS-DMEM/F12培养基正常培养24 h。1) Inoculate human endometrial mesenchymal stem cells at a density of 7000 cells/well in a 24-well plate with sterile small round slides. After the cells adhere to the wall, use 10% FBS-DMEM/ The F12 medium was cultured normally for 24 h.

2）吸弃培养基，室温下，PBS洗3次×5min。2) Discard the culture medium, and wash 3 times with PBS for 5 minutes at room temperature.

3）4%多聚甲醛室温固定30min。3) Fix with 4% paraformaldehyde at room temperature for 30 minutes.

4）弃多聚甲醛，室温下，PBS洗3次×5min。4) Discard the paraformaldehyde, wash with PBS 3 times × 5min at room temperature.

5）加入2N HCl，37℃水浴中作用10min。5) Add 2N HCl and act in a water bath at 37°C for 10 minutes.

6）加入0.1% Triton x-100-PBS 500μl，室温孵育5min。6) Add 500 μl of 0.1% Triton x-100-PBS and incubate at room temperature for 5 minutes.

7）室温下，PBS洗3次×5min。7) Wash 3 times with PBS for 5 minutes at room temperature.

8）10%山羊血清湿盒中室温封闭1h。8) Block for 1 hour at room temperature in a wet box with 10% goat serum.

9）BrdU一抗用PBS 1∶100稀释，湿盒中4℃孵育过夜。9) The BrdU primary antibody was diluted 1:100 with PBS, and incubated overnight at 4°C in a humid chamber.

10）室温下，PBS洗3次×5min。10) Wash with PBS 3 times for 5 minutes at room temperature.

11）TRITC标记山羊抗大鼠荧光二抗1∶100稀释，湿盒中37℃避光孵育1h。此步骤之后所有步骤均避光操作，尽量减少荧光淬灭。11) TRITC-labeled goat anti-rat fluorescent secondary antibody was diluted 1:100, and incubated in a humid chamber at 37°C for 1 hour in the dark. All steps after this step were performed in the dark to minimize fluorescence quenching.

12）室温下，PBS洗3次×5min。12) Wash with PBS 3 times for 5 minutes at room temperature.

13）加入DAPI工作液(1μg/ml)，湿盒中室温孵育10min。13) Add DAPI working solution (1 μg/ml), and incubate at room temperature for 10 minutes in a wet box.

14）室温下，PBS洗3次×5min。14) Wash with PBS 3 times for 5 minutes at room temperature.

15）干燥后，用抗荧光衰减封片剂封片。15) After drying, mount the slides with anti-fluorescence attenuation mounting medium.

16）荧光显微镜观察、摄片。16) Fluorescence microscope observation and filming.

图6中A和B显示了人子宫内膜间充质干细胞的增殖效果。A and B in Figure 6 show the proliferation effect of human endometrial mesenchymal stem cells.

以人骨髓间充质干细胞代替人子宫内膜间充质干细胞，重复上述试验过程，得到图6中C和D显示的人骨髓间充质干细胞增殖效果。Human bone marrow mesenchymal stem cells were used instead of human endometrial mesenchymal stem cells, and the above test process was repeated to obtain the proliferation effects of human bone marrow mesenchymal stem cells shown in C and D in FIG. 6 .

图6中，A和C是547nm波长激光激发TRITC染料得到的荧光图片，图中显示出的是BrdU阳性细胞，即处于增殖状态的细胞。B和D分别是在与A、C相同视野下，358nm波长激光激发下的荧光图片，显示所有细胞核。In Fig. 6, A and C are fluorescence pictures obtained by exciting TRITC dye with a 547nm wavelength laser, and the pictures show BrdU-positive cells, that is, cells in a proliferating state. B and D are the fluorescence pictures excited by 358nm wavelength laser under the same field of view as A and C respectively, showing all cell nuclei.

图中E的统计结果表示A与B的细胞数百分比、C与D的细胞数百分比，为BrdU阳性细胞率，反映细胞的增殖能力。The statistical results of E in the figure represent the percentage of cell number of A and B, and the percentage of cell number of C and D, which is the BrdU positive cell rate and reflects the proliferation ability of cells.

统计结果显示，人子宫内膜间充质干细胞具有增殖能力，在体外能够快速扩增，24h BrdU阳性细胞率84.67%±2.84%。而同批实验的相同年龄段供体来源、相同代数的人骨髓间充质干细胞的24h BrdU阳性率为70.67%±4.14%，二者比较，差异具有统计学意义(p＜0.05)。Statistical results show that human endometrial mesenchymal stem cells have the ability to proliferate and can rapidly expand in vitro, and the 24h BrdU-positive cell rate is 84.67%±2.84%. In the same batch of experiments, the 24h BrdU positive rate of human bone marrow mesenchymal stem cells from donors of the same age group and the same generation was 70.67%±4.14%, and the difference was statistically significant ( p <0.05).

上述试验结果说明，本发明成功分离培养获得的人子宫内膜间充质干细胞的增殖潜能明显高于人骨髓来源的间充质干细胞，即从人子宫内膜组织中分离得到的新型间充质干细胞具有较目前广泛应用的骨髓间充质干细胞更强的增殖能力。The above test results show that the proliferation potential of human endometrial mesenchymal stem cells successfully isolated and cultured in the present invention is significantly higher than that of human bone marrow-derived mesenchymal stem cells, that is, a new type of mesenchymal stem cells isolated from human endometrial tissue. Stem cells have a stronger proliferative ability than bone marrow mesenchymal stem cells, which are widely used at present.

综上所述，本发明两种分离培养方法均可以分离提取到人子宫内膜间充质干细胞。通过活细胞形态观察、细胞表面标志物检测，以及细胞迁移能力、体外诱导分化能力、增殖能力的检测，均证实本发明分离培养获得的人子宫内膜间充质干细胞不仅是一种新的来源的、具有功能的间充质干细胞，而且这种新的间充质干细胞具有更强的增殖能力，能够进行更快的体外扩增而获得足够数量的、活力旺盛的优质间充质干细胞，可以作为组织工程领域优质的种子细胞，靶向包括缺血性疾病、神经损伤、神经退行性疾病、年龄相关的退行性病变等在内的各种疾病进行细胞移植治疗。进而，本发明分离提取的人子宫内膜间充质干细胞还可以用于开发医学美容、亚健康人群保健、老年个体年轻化等方面的产品。In summary, the two isolation and culture methods of the present invention can isolate and extract human endometrial mesenchymal stem cells. Through the observation of living cell morphology, the detection of cell surface markers, and the detection of cell migration ability, in vitro induction differentiation ability, and proliferation ability, it is confirmed that the human endometrial mesenchymal stem cells obtained by the isolation and culture of the present invention are not only a new source Functional mesenchymal stem cells, and this new type of mesenchymal stem cells has stronger proliferation ability, and can be expanded faster in vitro to obtain a sufficient number of vigorous and high-quality mesenchymal stem cells. As a high-quality seed cell in the field of tissue engineering, it targets various diseases including ischemic disease, nerve injury, neurodegenerative disease, age-related degenerative disease, etc. for cell transplantation therapy. Furthermore, the human endometrial mesenchymal stem cells isolated and extracted in the present invention can also be used to develop products in aspects such as medical cosmetology, health care for sub-healthy people, and rejuvenation of elderly individuals.

Claims (10)

1. a kind of people's endometrial tissue derived mesenchymal stem cell with strong proliferative capacity, people's endometrial tissue comes Source mescenchymal stem cell is isolated from the endometrial tissue of people.

2. the isolated culture method of people's endometrial tissue derived mesenchymal stem cell, is to pass through enzymatic isolation method described in claim 1 It digests human endometrial to organize to obtain single cell suspension, is directly inoculated in mescenchymal stem cell culture medium and is cultivated and passed Generation purifying, obtains the mescenchymal stem cell.

3. the isolated culture method of people's endometrial tissue derived mesenchymal stem cell according to claim 2, feature It is to carry out as steps described below:

1) using the PBS buffer solution containing 250~350 μ g/ml clostridiopetidase As III and 30~50 μ g/ml DNase I as digestive juice, by institute It states digestive juice and is mixed with people's endometrial tissue according to 2~4: 1 volume ratio, after 37 DEG C of 40~60min of water-bath concussion digestion, taken out Digestion product supernatant is taken, 10vol% FBS-DMEM/F12 is added with 1: 1 volume ratio and is neutralized, is obtained after terminating digestion Single cell suspension；

2) single cell suspension is filtered with 200 mesh cell strainers, 1000~1200r/min centrifugation obtains cell mass；

3) 10vol% FBS-DMEM/F12 culture medium, 37 DEG C, 5%CO are added in the cell mass₂It is trained in cell incubator It supports, is no less than 1 secondary culture purifying, isolated people's endometrial tissue derived mesenchymal stem cell.

4. the isolated culture method of people's endometrial tissue derived mesenchymal stem cell, is to pass through enzymatic isolation method described in claim 1 It digests human endometrial to organize to obtain single cell suspension, isolated using density-gradient centrifugation method includes that mesenchyma is dry thin The cell mass of born of the same parents is inoculated in mescenchymal stem cell culture medium, is cultivated and passed on purifying, and it is dry thin to obtain the mesenchyma Born of the same parents.

5. the isolated culture method of people's endometrial tissue derived mesenchymal stem cell according to claim 4, feature It is to carry out as steps described below:

2) single cell suspension is filtered with 200 mesh cell strainers, 1000~1200r/min centrifugation obtains cell mass；

3) respectively prepare density be 1.048~1.052g/ml, three kinds of 1.058~1.062g/ml, 1.070~1.084g/ml Graded Density buffer containing Percoll and Ads is denoted as A liquid, B liquid and C liquid respectively, and wherein B liquid is with the phenol red mark of 0.01g/l Know；

4) isometric B liquid is moved into the centrifuge tube added with A liquid, gently insertion centrifugation bottom of the tube is slowly added dropwise below A liquid, Form the interface of two layers of liquid separation；

5) cell mass that step 2 obtains is resuspended with the C liquid isometric with A liquid, cell suspension is obtained, by the cell suspension Insertion centrifugation bottom of the tube is slowly added to below B liquid, and being formed has colourless-red-colourless clear separating interface of three-layer liquid body Graded Density buffer；

6) with 1300~1700r/min centrifugation, cell is dispersed in the different densities region of Graded Density buffer, draws B liquid The cell suspension of lower section and C liquid upper area, is put into the centrifuge tube for being previously added 15~30ml 10vol% FBS-DMEM/F12 In, it is soft to mix；

7) 1000~1300r/min centrifugation obtains cell mass, and 10~20ml 10vol% FBS-DMEM/F12 is added, soft mixed Even, 1000~1300r/min is centrifuged and collects cell mass；

8) cell mass is resuspended with 10vol%FBS-DMEM/F12 culture medium, be inoculated in culture bottle or culture dish, 37 DEG C, 5%CO₂ It is cultivated in cell incubator, is no less than 1 secondary culture purifying, isolated people's endometrial tissue derived mesenchymal is dry Cell.

6. the isolated culture method of people's endometrial tissue derived mesenchymal stem cell according to claim 3 or 5, special Sign is that used people's endometrial tissue is the tissue cleaned up with PBS, and sufficiently shred.

7. the isolated culture method of people's endometrial tissue derived mesenchymal stem cell according to claim 3 or 5, special Sign is that people's endometrial tissue is merged the cell suspension that digestion obtains with digestive juice digestion no less than 2 times.

8. people's endometrial tissue derived mesenchymal stem cell described in claim 1 is in preparation prevention or treats ischemic disease Application in drug.

9. people's endometrial tissue derived mesenchymal stem cell described in claim 1 is in preparation prevention or treatment nervus retrogression disease Application in the drug of disease or other age-dependent degenerative diseases.

10. people's endometrial tissue derived mesenchymal stem cell described in claim 1 is protected in preparation medical cosmetology, sub-health population Application in strong, older individuals rejuvenation product.

CN201810930181.6A 2018-08-15 2018-08-15 The isolated culture method of people's endometrial tissue derived mesenchymal stem cell Pending CN108998411A (en)

Priority Applications (1)

Application Number	Priority Date	Filing Date	Title
CN201810930181.6A CN108998411A (en)	2018-08-15	2018-08-15	The isolated culture method of people's endometrial tissue derived mesenchymal stem cell

Applications Claiming Priority (1)

Application Number	Priority Date	Filing Date	Title
CN201810930181.6A CN108998411A (en)	2018-08-15	2018-08-15	The isolated culture method of people's endometrial tissue derived mesenchymal stem cell

Publications (1)

Publication Number	Publication Date
CN108998411A true CN108998411A (en)	2018-12-14

Family

ID=64592696

Family Applications (1)

Application Number	Title	Priority Date	Filing Date
CN201810930181.6A Pending CN108998411A (en)	2018-08-15	2018-08-15	The isolated culture method of people's endometrial tissue derived mesenchymal stem cell