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CN108295311A - A kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel - Google Patents

  • ️Fri Jul 20 2018
A kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel Download PDF

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Publication number
CN108295311A
CN108295311A CN201810186905.0A CN201810186905A CN108295311A CN 108295311 A CN108295311 A CN 108295311A CN 201810186905 A CN201810186905 A CN 201810186905A CN 108295311 A CN108295311 A CN 108295311A Authority
CN
China
Prior art keywords
kidney
preparation
ecm
extracellular matrix
temperature sensitive
Prior art date
2018-03-07
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
CN201810186905.0A
Other languages
Chinese (zh)
Inventor
贾瑞鹏
周六化
葛余正
周昌成
许露伟
吴然
赵峰
徐忠乐
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.)
Nanjing First Hospital
Original Assignee
Nanjing First Hospital
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.)
2018-03-07
Filing date
2018-03-07
Publication date
2018-07-20
2018-03-07 Application filed by Nanjing First Hospital filed Critical Nanjing First Hospital
2018-03-07 Priority to CN201810186905.0A priority Critical patent/CN108295311A/en
2018-07-20 Publication of CN108295311A publication Critical patent/CN108295311A/en
Status Pending legal-status Critical Current

Links

  • 210000003734 kidney Anatomy 0.000 title claims abstract description 108
  • 102000010834 Extracellular Matrix Proteins Human genes 0.000 title claims abstract description 61
  • 108010037362 Extracellular Matrix Proteins Proteins 0.000 title claims abstract description 61
  • 210000002744 extracellular matrix Anatomy 0.000 title claims abstract description 61
  • 238000002360 preparation method Methods 0.000 title claims abstract description 58
  • 239000000017 hydrogel Substances 0.000 title claims abstract description 47
  • 230000001954 sterilising effect Effects 0.000 claims abstract description 22
  • 239000007788 liquid Substances 0.000 claims abstract description 21
  • 102000007260 Deoxyribonuclease I Human genes 0.000 claims abstract description 19
  • 108010008532 Deoxyribonuclease I Proteins 0.000 claims abstract description 19
  • 210000004027 cell Anatomy 0.000 claims abstract description 16
  • 230000029087 digestion Effects 0.000 claims abstract description 16
  • 238000003756 stirring Methods 0.000 claims abstract description 8
  • 210000004204 blood vessel Anatomy 0.000 claims abstract description 7
  • 230000018044 dehydration Effects 0.000 claims abstract description 6
  • 238000006297 dehydration reaction Methods 0.000 claims abstract description 6
  • 238000001802 infusion Methods 0.000 claims abstract description 6
  • 229920003023 plastic Polymers 0.000 claims abstract description 6
  • 230000010412 perfusion Effects 0.000 claims abstract description 4
  • 239000000243 solution Substances 0.000 claims description 18
  • 229920004890 Triton X-100 Polymers 0.000 claims description 16
  • 239000013504 Triton X-100 Substances 0.000 claims description 16
  • HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
  • XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
  • VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
  • FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
  • 239000012153 distilled water Substances 0.000 claims description 10
  • 238000000034 method Methods 0.000 claims description 7
  • 235000013399 edible fruits Nutrition 0.000 claims description 6
  • UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
  • IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 claims description 5
  • 102000057297 Pepsin A Human genes 0.000 claims description 5
  • 108090000284 Pepsin A Proteins 0.000 claims description 5
  • 239000007983 Tris buffer Substances 0.000 claims description 5
  • 210000004369 blood Anatomy 0.000 claims description 5
  • 239000008280 blood Substances 0.000 claims description 5
  • 239000001110 calcium chloride Substances 0.000 claims description 5
  • 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
  • 235000011148 calcium chloride Nutrition 0.000 claims description 5
  • 239000000470 constituent Substances 0.000 claims description 5
  • 238000001914 filtration Methods 0.000 claims description 5
  • 238000011010 flushing procedure Methods 0.000 claims description 5
  • 230000002262 irrigation Effects 0.000 claims description 5
  • 238000003973 irrigation Methods 0.000 claims description 5
  • 229940111202 pepsin Drugs 0.000 claims description 5
  • 230000008569 process Effects 0.000 claims description 5
  • 239000011780 sodium chloride Substances 0.000 claims description 5
  • LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 5
  • GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 5
  • 241000894006 Bacteria Species 0.000 claims 1
  • 238000002347 injection Methods 0.000 abstract description 6
  • 239000007924 injection Substances 0.000 abstract description 6
  • 239000004615 ingredient Substances 0.000 abstract description 4
  • 230000000975 bioactive effect Effects 0.000 abstract description 2
  • 230000031018 biological processes and functions Effects 0.000 abstract description 2
  • 230000001413 cellular effect Effects 0.000 abstract description 2
  • 210000005084 renal tissue Anatomy 0.000 abstract description 2
  • 229920002113 octoxynol Polymers 0.000 abstract 1
  • 210000000130 stem cell Anatomy 0.000 description 10
  • 238000011049 filling Methods 0.000 description 6
  • 230000006378 damage Effects 0.000 description 3
  • 230000008014 freezing Effects 0.000 description 3
  • 238000007710 freezing Methods 0.000 description 3
  • 210000000056 organ Anatomy 0.000 description 3
  • 230000008439 repair process Effects 0.000 description 3
  • 230000004083 survival effect Effects 0.000 description 3
  • 238000002054 transplantation Methods 0.000 description 3
  • 230000000694 effects Effects 0.000 description 2
  • 210000005259 peripheral blood Anatomy 0.000 description 2
  • 239000011886 peripheral blood Substances 0.000 description 2
  • 206010067484 Adverse reaction Diseases 0.000 description 1
  • 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 1
  • 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
  • 229920002683 Glycosaminoglycan Polymers 0.000 description 1
  • 206010061481 Renal injury Diseases 0.000 description 1
  • 208000027418 Wounds and injury Diseases 0.000 description 1
  • 239000004480 active ingredient Substances 0.000 description 1
  • 230000006838 adverse reaction Effects 0.000 description 1
  • 230000004520 agglutination Effects 0.000 description 1
  • 230000008901 benefit Effects 0.000 description 1
  • 230000017531 blood circulation Effects 0.000 description 1
  • 230000036760 body temperature Effects 0.000 description 1
  • 239000003795 chemical substances by application Substances 0.000 description 1
  • 150000001875 compounds Chemical class 0.000 description 1
  • 238000010586 diagram Methods 0.000 description 1
  • 238000010828 elution Methods 0.000 description 1
  • 239000003102 growth factor Substances 0.000 description 1
  • 238000007654 immersion Methods 0.000 description 1
  • 238000002513 implantation Methods 0.000 description 1
  • 230000006872 improvement Effects 0.000 description 1
  • 208000014674 injury Diseases 0.000 description 1
  • 210000003292 kidney cell Anatomy 0.000 description 1
  • 208000037806 kidney injury Diseases 0.000 description 1
  • 230000002045 lasting effect Effects 0.000 description 1
  • 230000014759 maintenance of location Effects 0.000 description 1
  • 238000002156 mixing Methods 0.000 description 1
  • 239000000203 mixture Substances 0.000 description 1
  • 230000004048 modification Effects 0.000 description 1
  • 238000012986 modification Methods 0.000 description 1
  • 230000036542 oxidative stress Effects 0.000 description 1
  • 230000003076 paracrine Effects 0.000 description 1
  • 238000011476 stem cell transplantation Methods 0.000 description 1
  • 238000013268 sustained release Methods 0.000 description 1
  • 239000012730 sustained-release form Substances 0.000 description 1
  • 230000009885 systemic effect Effects 0.000 description 1
  • 210000001519 tissue Anatomy 0.000 description 1

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3633—Extracellular matrix [ECM]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/26—Materials or treatment for tissue regeneration for kidney reconstruction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Botany (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Dispersion Chemistry (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Materials For Medical Uses (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

The invention discloses a kind of preparation methods of temperature sensitive type extracellular matrix of kidney hydrogel, including preparation process in detail below：Kidney preliminary treatment, kidney take off Cell infusion, kidney rinses, dehydration sterilizing, stir digestion, plastic.The present invention sets pipe by the arteria renalis using perfusion and is rinsed de- cell, and according to the distribution of blood vessel, de-cell liquid can reach almost everywhere renal tissue so that de- cell is more abundant.And the ingredient after cell rupture can be flowed with liquid, discharge in time, de- cell is more efficient, greatly reduce the residual of possible cellular content, and use Triton X 100 and Type I DNase I combined injections, both nuclear fraction was fully eluted, and also remained the bioactive ingredients of kidney ECM to greatest extent, better biological action can be played.

Description

A kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel

Technical field

The present invention relates to hydrogel preparing technical field, more particularly to a kind of temperature sensitive type extracellular matrix of kidney hydrogel Preparation method.

Background technology

Stem cell transplantation has become a kind of important means of kidney injury reparation, includes mainly two ways：Through peripheral blood Pipe injection transplantation and local injection transplantation.It is although easy to operate through peripheral blood vessel injection transplantation, but the dry of target organ can be reached Cell quantity is less, thereby increases and it is possible to lead to systemic adverse reactions；Although local injection transplanting can guarantee higher target organ stem cell Implantation rate, but the survival rate of stem cell is relatively low, and part can remove target organ with blood flow

Extracellular matrix (ECM) hydrogel is remarkably improved the retention rate and survival rate of stem cell, enhances the group of stem cell Knit repair ability.But existing extracellular matrix preparation method elutes insufficient more or damages larger, and kidney source property to ECM The preparation of ECM hydrogels, is not reported still at present.Therefore, a kind of preparation of temperature sensitive type extracellular matrix of kidney hydrogel is invented Method is necessary to solve the above problems.

Invention content

The purpose of the present invention is to provide a kind of preparation methods of temperature sensitive type extracellular matrix of kidney hydrogel, in solution State the problem of being proposed in background technology.

To achieve the above object, the present invention provides the following technical solutions：A kind of temperature sensitive type extracellular matrix of kidney hydrogel Preparation method, including preparation process in detail below：

One, the preparation of kidney ECM：

Step 1：Kidney preliminary treatment：Animal kidney is taken, retains kidney base of a fruit structure, and carry out the arteria renalis and set pipe, then gives 2h is rinsed with 500ml distilled water quick fillings, to remove remaining blood constituent in renal blood vessels；

Step 2：Kidney takes off Cell infusion：After flushing, the Triton X-100 quick fillings 4h of 1000ml is utilized Afterwards, then with the Triton X-100 of 1000ml continue that 12h slowly is perfused, after the completion of Triton X-100 perfusions, utilize 50U/ Ml Type I DNase I liquid quick fillings 4h；

Step 3：Kidney rinses：After rinsing, with distilled water irrigation 2h, remnants Triton in kidney are removed X-100 and Type I DNase I liquid；

Two, the preparation of kidney ECM hydrogels：

Step 1：Dehydration sterilizing：After the preparation of kidney ECM is completed, kidney is sloughed into moisture in freeze drier, And it shreds in powdered, while importing ethylene oxide gas and sterilizing；

Step 2：Powdered kidney ECM is dissolved in the 0.1M hydrochloric acid containing pepsin, and is stirred at ambient temperature Mix digestion；

Step 3：Plastic：After step 2 is completed, the sterilizing 10xPBS of 1/9 overall solution volume, and profit is inwardly added It is 15mg/ml to adjust final ECM hydrogel concentrations with sterilizing 1xPBS.

Preferably, animal kidney is fresh kidney, and the take-off time out of animal body in the step of preparation of kidney ECM one For 10min~60min.

Preferably, Triton X-100 concentration is set as 0.5%~1.5%, 50U/ in the step of preparation of kidney ECM two Ml Type I DNase I liquid be by 10mM Tris-base, 2mM MgCl2,2mM CaCl2 and 150mM NaCl mixing and At, and Type I DNase I liquid pH value is in 7.6.

Preferably, in the preparation process one of kidney ECM hydrogels freeze drier temperature be maintained at -30 DEG C~-50 DEG C it Between.

Preferably, digestion time is stirred in the preparation process two of kidney ECM hydrogels and is set as 48h, and is digested and terminated Afterwards, the pH value of NaOH adjustment solution of 1M is added to 7.4.

Preferably, when the preparation process three of kidney ECM hydrogels carries out, it need to remain and carry out on ice.

Preferably, when carrying out, whole flow process is required for super for the preparation of kidney ECM and the preparation of kidney ECM hydrogels The solution completed in net platform, and be added has to pass through sterilizing or biofilter filtering.

The technique effect and advantage of the present invention：

1, the present invention sets pipe by the arteria renalis using perfusion and is rinsed de- cell, according to the distribution of blood vessel, takes off cell Liquid energy enough reaches almost everywhere renal tissue so that de- cell is more abundant.And the ingredient after cell rupture can be with liquid Flowing is discharged in time, and de- cell is more efficient, greatly reduces the residual of possible cellular content；

2, the present invention uses Triton X-100 and Type I DNase I combined injections, is both filled to nuclear fraction Divide elution, also remains the bioactive ingredients of kidney ECM to greatest extent, better biological action can be played；

3, kidney ECM hydrogels of the invention include collagenous fibres, and (37 DEG C or so) can be rapid under body temperature environment Become agglutination, form crisscross porous structure, is conducive to stem cell immersion, and the loss of stem cell can be prevented, and kidney Dirty ECM hydrogels can mitigate oxidativestress damage, improve the survival rate of stem cell；

4, the paracrine ability of stem cell can be improved in kidney ECM hydrogels of the invention, compound compared with simple stem cell The stem cell of hydrogel can secrete more bioactie agents, enhance the repair ability to injury tissue；

5, can have with growth factors such as bFGF, HGF rich in sulfated glycosaminoglycan etc. in kidney ECM hydrogels of the invention Effect combines, and has the characteristics that sustained release, can play lasting repair.

Description of the drawings

Fig. 1 is the preparation process schematic diagram of the present invention.

Specific implementation mode

Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other Embodiment shall fall within the protection scope of the present invention.

Embodiment 1：A kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel, including step is prepared in detail below Suddenly：

One, the preparation of kidney ECM：

Step 1：Kidney preliminary treatment：Take the fresh kidney of animal, and out of animal body take-off time be 10min~ 60min retains kidney base of a fruit structure, and carries out the arteria renalis and set pipe, then gives 500ml distilled water quick fillings and rinses 2h, to go Except remaining blood constituent in renal blood vessels；

Step 2：Kidney takes off Cell infusion：After flushing, a concentration of 0.5% 1000mlTriton X- are utilized After 100 quick filling 4h, then continued with the Triton X-100 of 1000ml that 12h slowly is perfused, when Triton X-100 have been perfused Cheng Hou, the 50U/ml Type mixed using 10mM Tris-base, 2mM MgCl2,2mM CaCl2 and 150mM NaCl I DNase I liquid quick filling 4h, and Type I DNase I liquid pH value is in 7.6；

Step 3：Kidney rinses：After rinsing, with distilled water irrigation 2h, remnants Triton in kidney are removed X-100 and Type I DNase I liquid；

Two, the preparation of kidney ECM hydrogels：

Step 1：Dehydration sterilizing：After the preparation of kidney ECM is completed, the freezing by kidney at -30 DEG C~-50 DEG C is done Moisture is sloughed in dry machine, and is shredded in powdered, while being imported ethylene oxide gas and being sterilized；

Step 2：Stirring digestion：Powdered kidney ECM is dissolved in the 0.1M hydrochloric acid containing pepsin, and in room The pH value of NaOH adjustment solution of 1M is added to 7.4 after digestion in stirring digestion 48h under the conditions of temperature；

Step 3：Plastic：After step 2 is completed, the sterilizing 10xPBS of 1/9 overall solution volume, and profit is inwardly added Adjusting final ECM hydrogel concentrations with sterilizing 1xPBS, to be 15mg/ml need to remain and carry out on ice when step 3 carries out；

When carrying out, whole flow process is required for complete in super-clean bench for the preparation of kidney ECM and the preparation of kidney ECM hydrogels At, and the solution being added has to pass through sterilizing or biofilter filtering.

Embodiment 2：A kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel, including step is prepared in detail below Suddenly：

One, the preparation of kidney ECM：

Step 1：Kidney preliminary treatment：Take the fresh kidney of animal, and out of animal body take-off time be 10min~ 60min retains kidney base of a fruit structure, and carries out the arteria renalis and set pipe, then gives 500ml distilled water quick fillings and rinses 2h, to go Except remaining blood constituent in renal blood vessels；

Step 2：Kidney takes off Cell infusion：After flushing, a concentration of 1.0% 1000mlTriton X- are utilized After 100 quick filling 4h, then continued with the Triton X-100 of 1000ml that 12h slowly is perfused, when Triton X-100 have been perfused Cheng Hou, the 50U/ml Type mixed using 10mM Tris-base, 2mM MgCl2,2mM CaCl2 and 150mM NaCl I DNase I liquid quick filling 4h, and Type I DNase I liquid pH value is in 7.6；

Step 3：Kidney rinses：After rinsing, with distilled water irrigation 2h, remnants Triton in kidney are removed X-100 and Type I DNase I liquid；

Two, the preparation of kidney ECM hydrogels：

Step 1：Dehydration sterilizing：After the preparation of kidney ECM is completed, the freezing by kidney at -30 DEG C~-50 DEG C is done Moisture is sloughed in dry machine, and is shredded in powdered, while being imported ethylene oxide gas and being sterilized；

Step 2：Stirring digestion：Powdered kidney ECM is dissolved in the 0.1M hydrochloric acid containing pepsin, and in room The pH value of NaOH adjustment solution of 1M is added to 7.4 after digestion in stirring digestion 48h under the conditions of temperature；

Step 3：Plastic：After step 2 is completed, the sterilizing 10xPBS of 1/9 overall solution volume, and profit is inwardly added Adjusting final ECM hydrogel concentrations with sterilizing 1xPBS, to be 15mg/ml need to remain and carry out on ice when step 3 carries out；

When carrying out, whole flow process is required for complete in super-clean bench for the preparation of kidney ECM and the preparation of kidney ECM hydrogels At, and the solution being added has to pass through sterilizing or biofilter filtering.

Embodiment 3：A kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel, including step is prepared in detail below Suddenly：

One, the preparation of kidney ECM：

Step 1：Kidney preliminary treatment：Take the fresh kidney of animal, and out of animal body take-off time be 10min~ 60min retains kidney base of a fruit structure, and carries out the arteria renalis and set pipe, then gives 500ml distilled water quick fillings and rinses 2h, to go Except remaining blood constituent in renal blood vessels；

Step 2：Kidney takes off Cell infusion：After flushing, a concentration of 1.5% 1000mlTriton X- are utilized After 100 quick filling 4h, then continued with the Triton X-100 of 1000ml that 12h slowly is perfused, when Triton X-100 have been perfused Cheng Hou, the 50U/ml Type mixed using 10mM Tris-base, 2mM MgCl2,2mM CaCl2 and 150mM NaCl I DNase I liquid quick filling 4h, and Type I DNase I liquid pH value is in 7.6；

Step 3：Kidney rinses：After rinsing, with distilled water irrigation 2h, remnants Triton in kidney are removed X-100 and Type I DNase I liquid；

Two, the preparation of kidney ECM hydrogels：

Step 1：Dehydration sterilizing：After the preparation of kidney ECM is completed, the freezing by kidney at -30 DEG C~-50 DEG C is done Moisture is sloughed in dry machine, and is shredded in powdered, while being imported ethylene oxide gas and being sterilized；

Step 2：Stirring digestion：Powdered kidney ECM is dissolved in the 0.1M hydrochloric acid containing pepsin, and in room The pH value of NaOH adjustment solution of 1M is added to 7.4 after digestion in stirring digestion 48h under the conditions of temperature；

Step 3：Plastic：After step 2 is completed, the sterilizing 10xPBS of 1/9 overall solution volume, and profit is inwardly added Adjusting final ECM hydrogel concentrations with sterilizing 1xPBS, to be 15mg/ml need to remain and carry out on ice when step 3 carries out；

When carrying out, whole flow process is required for complete in super-clean bench for the preparation of kidney ECM and the preparation of kidney ECM hydrogels At, and the solution being added has to pass through sterilizing or biofilter filtering.

Following data can be obtained by Examples 1 to 3

By above table data it is found that when Triton X-100 it is a concentration of 1.0% when, to animal kidney cell elute imitate Fruit is more abundant, and destroys minimum to animal kidney active ingredient, to ensure that kidney ECM hydrogel quality obtained reaches It is optimal.

Finally it should be noted that：The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used With technical scheme described in the above embodiments is modified or equivalent replacement of some of the technical features, All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in the present invention's Within protection domain.

Claims (7)

1. a kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel, which is characterized in that including preparing step in detail below Suddenly：

One, the preparation of kidney ECM：

Step 1：Kidney preliminary treatment：Animal kidney is taken, retains kidney base of a fruit structure, and carry out the arteria renalis and set pipe, then gives 500ml distilled water quick fillings rinse 2h, to remove remaining blood constituent in renal blood vessels；

Step 2：Kidney takes off Cell infusion：After flushing, after the Triton X-100 quick fillings 4h of 1000ml, Continued that 12h slowly is perfused with the Triton X-100 of 1000ml again, after the completion of Triton X-100 perfusions, utilizes 50 U/ml Type I DNase I liquid quick fillings 4h；

Step 3：Kidney rinses：After rinsing, with distilled water irrigation 2h, remnants Triton X- in kidney are removed 100 and Type I DNase I liquid；

Two, the preparation of kidney ECM hydrogels：

Step 1：Dehydration sterilizing：After the preparation of kidney ECM is completed, kidney is sloughed into moisture in freeze drier, and It is in powdered to shred, while importing ethylene oxide gas and sterilizing；

Step 2：Stirring digestion：Powdered kidney ECM is dissolved in the 0.1M hydrochloric acid containing pepsin, and in room temperature item Digestion is stirred under part；

Step 3：Plastic：After step 2 is completed, the sterilizing 10xPBS of 1/9 overall solution volume is inwardly added, and utilize and go out It is 15mg/ml that bacterium 1xPBS, which adjusts final ECM hydrogel concentrations,.

2. a kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel according to claim 1, it is characterised in that： Animal kidney is fresh kidney in the step of preparation of kidney ECM one, and take-off time is 10min ~ 60min out of animal body.

3. a kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel according to claim 1, it is characterised in that： The preparation of kidney ECM is that Triton X-100 concentration is set as 0.5% ~ 1.5% in step 2,50 U/ml Type I DNase I Liquid is mixed by 10mM Tris-base, 2mM MgCl2,2mM CaCl2 and 150mM NaCl, and Type I DNase I Liquid pH value is in 7.6.

4. a kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel according to claim 1, it is characterised in that： Freeze drier temperature is maintained between -30 DEG C ~ -50 DEG C in the preparation process one of kidney ECM hydrogels.

5. a kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel according to claim 1, it is characterised in that： Digestion time is stirred in the preparation process two of kidney ECM hydrogels and is set as 48h, and after digestion, the NaOH of 1M is added The pH value of solution is adjusted to 7.4.

6. a kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel according to claim 1, it is characterised in that： When the preparation process three of kidney ECM hydrogels carries out, it need to remain and carry out on ice.

7. a kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel according to claim 1, it is characterised in that： When carrying out, whole flow process is required for completing in super-clean bench for the preparation of kidney ECM and the preparation of kidney ECM hydrogels, and The solution of addition has to pass through sterilizing or biofilter filtering.

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