CN105854084A - Extracellular matrix biomaterial for resisting cell premature senility and preparation method and application thereof - Google Patents

Extracellular matrix biomaterial for resisting cell premature senility and preparation method and application thereof Download PDF

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CN105854084A
CN105854084A CN201610202228.8A CN201610202228A CN105854084A CN 105854084 A CN105854084 A CN 105854084A CN 201610202228 A CN201610202228 A CN 201610202228A CN 105854084 A CN105854084 A CN 105854084A
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cell
extracellular matrix
early ageing
mscs
matrix biomaterial
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何帆
周龙
陈曦
张文
罗宗平
杨惠林
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First Affiliated Hospital of Suzhou University
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First Affiliated Hospital of Suzhou University
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    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • 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
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action

Abstract

The present invention discloses an extracellular matrix biomaterial for resisting cell premature senility and a preparation method and application thereof. The matrix is secreted from culture of umbilical cord mesenchymal stem cells and is resistant to cell premature senility. The umbilical cord mesenchymal stem cells are cultured in a complete medium; when the cell density is close to 90%, a complete culture solution containing ascorbic acid is added, and culture is carried out for 7-10 days; then a cell removal solution is added to construct an extracellular matrix biomaterial. The umbilical cord mesenchymal stem cells are obtained from human source, swine source, murine source or rabbit source. The extracellular matrix biomaterial for resisting cell premature senility can be applied to cell culture, including cell transplantation therapy, regenerative medicine and tissue engineering, etc. Studies show that the intracellular MSCs cultured in ECM environment has ROS content significantly decreased, which confirms that ECM from UC-MSCs can alleviate the UC-MSCs premature senility induced by H2O2 and provide a new policy to address the challenges brought by cells aging.

Description

A kind of extracellular matrix biomaterial of anti-cell early ageing and its preparation method and application
Technical field
The invention belongs to the technical field of biological materials of organizational project, be specifically related to the extracellular base of a kind of anti-cell early ageing Matter biomaterial and its preparation method and application.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSCs) can obtain from multiple adult's tissue, by There is the compatible feature of self, Multidirectional Differentiation and immunity in the regenerative medicine theoretical based on cell and tissue work in MSCs Journey aspect causes the extensive concern of clinic.MSCs autologous is derived mainly from bone marrow matrix, adipose tissue, synovial tissue and skin Skin tissue.MSCs obtains convenient from these tissues, is not related to problems of morals principles, it is simple to Clinical and experimental study.Recently, MSCs derives from and organizes outside embryo, holds two arteria umbilicalis and a umbilical vein as derived from Wharton's jelly(umbilical cord Glutinous albumen sample connective tissue) Human Umbilical Cord's mescenchymal stem cell (human umbilical cord-derived MSCs, UC-MSCs), Application comparison clinically has superiority.UC-MSCs can obtain by the way of Noninvasive, meets ethics road The requirement of moral.The multiplication capacity of the MSCs obtained compared to its hetero-organization, UC-MSCs is higher, has similar to embryonic stem cell Gene expression profile, subculture in vitro separately the most just there will be replicative senescence.
Similar with other body cells, MSCs breed in vitro occur growth retardation to enter before cellular senescence process having certain Life-span limit.Become in form during cell ageing is roomy and flat, even if basic the oldest of cell is forever stagnated in cell proliferation Metabolic capability still suffers from.MSCs is the oldest and the most feeble, and multi-lineage potential reduces, and limits its application in terms of clinical treatment.End Grain shortens, and DNA is impaired, oxidative stress, and Aging-associated gene expression all can cause cell ageing.The accumulation of active oxygen (reactive oxygen species, ROS), such as hydrogen peroxide (hydrogen peroxide, H2O2), cause oxidation to answer Swash, and then induction cell early ageing.The ROS of harmful level not only causes DNA oxidation to damage in the articular cartilage in Osteoarthritis Wound, also makes the regeneration potential of MSC be suppressed.
Cell ageing result in cell cycle irreversible stagnation, p16INK4αIt it is a kind of cyclin enzyme level Agent, the process of controllable cell cycle, by cell cycle regulation kinases regulation cell from the G1 phase to the S phase.
About the molecular mechanism existing numerous studies report of stem cell aging, generally believe with stem cell from internal to external Residing microenvironment changes relevant.MSCs once leaves external special microenvironment, gradually loses its self and multidirectional point The potential changed.The important part of stem cell microenvironment is ECM, and ECM maintains with biochemistry and the physical signalling of its complexity Cell function.The microenvironment that researchers are natural in having attempted to reconstituted cell, by using the combination of different ECM protein to adjust The destiny of control MSCs.Linsley etc. compare that fibronectin is former, type i collagen, three kinds of different ECM protein of fibronectin, find fibre Even proteinogen plays most important functions in terms of promoting MSCs Osteoblast Differentiation.But, natural ECM not only comprises various Protein, such as I, II, III, IV, V and VI Collagen Type VI, the attachment proteins such as fibronectin and laminin and macromolecule egg White glycan, and exist for cell and place is provided, strengthen the reaction that mechanical signal is stimulated by MSCs, as tension and the thorn of pressure Swashing, this is the interaction of the integrin-extracellular matrix by specific cell surface.Therefore in n cell origin ECM and have the cell cultivated in the matrix of ECM protein coating to show different qualities, but natural ECM regulation and control MSCs function Precise mechanism needs exploratory development further.
Recently, studies have reported that the de-cell ECM originating from MSCs is of value to expanding in a large number and multidirectional point of external MSCs Change potential.Our research indicate that de-cell ECM not only increases the biological antioxidant function of MSCs and strengthens before The characteristic of the anti-oxidant and self-bone grafting of intracellular epiphysin, and derive from the ECM of stem cell to the effect of the early ageing of MSCs and tune Control the potential mechanism of its early ageing to there is presently no and illustrate completely.
Summary of the invention
Present invention aims to MSCs expand in vitro and be inevitably exposed to phase in internal migration process The problem causing cell early ageing under big oxidative stress, it is provided that the extracellular matrix biomaterial of a kind of anti-cell early ageing, And the method for this extracellular matrix biomaterial (ECM) anti-cell early ageing and application.
To achieve these goals, the technical solution used in the present invention is:
The extracellular matrix biomaterial of anti-cell early ageing, it is the matrix cultivating umbilical cord mesenchymal stem cells secretion, this matrix Can the early ageing of anti-cell.The ECM prepared by umbilical cord mesenchymal stem cells secretion matrix, is had multiple proteins composition, can lead to Cross reduction SA-β-Ga activity, promote that cell enters the S phase, reduce intracellular ROS level, suppress p16INK4αGene and the expression of albumen Reach the purpose of anti-stem cell early ageing.
Fill stem cell between described umbilical cord and take from people source, pig source, mouse source or rabbit source.
The preparation method of the extracellular matrix biomaterial of described anti-cell early ageing, comprises the steps:
1) umbilical cord mesenchymal stem cells is cultivated in complete medium, when cell density is close to 90%, add containing Vitamin C The complete culture solution of acid is cultivated 7-10 days；
2) add de-cell liquid, PBS, obtain extracellular matrix biomaterial.
Described umbilical cord mesenchymal stem cells takes from people source, pig source, mouse source or rabbit source.
Described complete medium be component be α-MEM 80 ~ 90% volume ratio, hyclone 10 ~ 20% volume ratio, antibiosis Element 10 ~ 200 U/mL.
In described complete culture solution, the concentration of ascorbic acid is 100-200 μM.
Described de-cell liquid is formed by the phosphate buffered saline of pH7.4, containing Triton X-100 0.5 ~ 5% body Long-pending ratio and ammoniacal liquor 10-40mM.
The extracellular matrix biomaterial of the anti-cell early ageing that the present invention provides can be used for the cultivation of cell, moves including cell Plant treatment, the field such as regenerative medicine and organizational project.
When cell is cultivated, cell to be cultivated is added directly in the extracellular matrix biomaterial of the present invention, can prolong The aging of slow cell.
The application in preparing cosmetics, medicine or the health products that anti-cell is old and feeble of the biomaterial of the present invention.To this The biomaterial of invention and this area customary adjuvant are prepared by mixing into used for cosmetic in the aging delaying cell.
The present invention builds and obtains ECM and investigated H2O2UC-MSCs early ageing situation under You Dao, particularly as follows:
(A) umbilical cord mesenchymal stem cells (umbilical cord-derived mesenchymal stem cells,
UC-MSCs) in complete medium, (nutrient media components is α-MEM 80 ~ 90% volume ratio, hyclone 10 ~ 20% in cultivation Volume ratio, antibiotic 10 ~ 200 U/mL), when density is close to 90%, add the cultivation completely containing ascorbic acid (100-200uM) Liquid is cultivated 7-10 days, within every 3 days, changes liquid once, adds de-cell liquid afterwards and (by the phosphate buffered saline of pH7.4, contains Triton X-100 0.5 ~ 5% volume ratio and ammoniacal liquor 10-40mM), build and obtain ECM.
(B) UC-MSCs cultivates and is covered with in the orifice plate (ECM plate) of ECM at common orifice plate (TCPS plate) and bottom, and density connects When nearly 50%, add variable concentrations gradient H2O2 (50 μMs, 100 μMs, 200 μMs) process 2-4h, cause stem cell oxidation to answer Swash.(B) H is removed2O2The culture medium of rear addition serum-free cleans twice, after adding above-mentioned complete medium continuation cultivation 3-5 days Detection early ageing stem cell SA-β-Gal stained positive rate, intracellular ROS expression, G0/G1 phase ratio, cell in the cell cycle Interior p16INK4αGene and protein expression level.
We have employed the H of middle variable concentrations gradient2O2(50 μMs, 100 μMs, 200 μMs) produce oxidative stress environment Induction UC-MSCs early ageing.ECM plate is compared with TCPS plate, and H can effectively be restrained by ECM plate2O2The UC-MSCs early ageing of induction, especially H2O2When concentration is 50 μMs and 100 μMs.ECM can substantially alleviate cell early ageing, weakens aging-related phenotype, including SA-β-Gal Activity, ROS level, cell cycle distribution, p16INK4αContent.And, work as H2O2When concentration reaches 200uM, ECM can not stop The early ageing of MSCs.Therefore, ECM can alleviate the early ageing of MSCs under the oxidative stress of appropriate level, and the oxidation of unusual high levels should Swash and exceed cell maximum tolerance range, ultimately result in the cell ageing of irreversibility.
Transplant MSCs and in the application of clinical treatment, there are the biggest potentiality for repairing damaged tissues and the old and feeble organ of replacement, But pathological conditions in damaged tissue region and the oxidative stress along with abnormal level, will be one to choose greatly application clinically War.The ROS of abnormal level can cause the DNA damage of the MSCs of CFU-GM and transplanting, and DNA damage does not has appropriate reparation to be to lead Cause the principal element of cell ageing.MSCs in order to
The potential maintaining self must pull against the aging of oxidative stress induction.
MSCs extensively applies in cell transplantation, regenerative medicine and organizational project, but it is answered by oxidation inevitably Exciting raw aging, significantly limit its application, therefore opposing cell ageing is particularly important.Present invention demonstrates and derive from The ECM of UC-MSCs can alleviate H2O2The UC-MSCs early ageing of induction, this is that the challenge that solution cell ageing brings provides one New strategy, is beneficial to the MSCs application at clinicing aspect.
Beneficial effect:
The extracellular matrix biomaterial of the anti-cell early ageing that the present invention provides, secretes matrix system by umbilical cord mesenchymal stem cells Standby ECM, has multiple proteins composition, can promote that cell enters the S phase by reducing SA-β-Ga activity, reduce intracellular ROS Level, suppresses p16INK4αThe expression of gene and albumen reaches the purpose of anti-stem cell early ageing, and present invention research shows that MSCs trains Support the content of intracellular ROS in the environment of ECM to be decreased obviously it was confirmed the ECM deriving from UC-MSCs can alleviate H2O2Induction UC-MSCs early ageing, this is to solve the challenge that brings of cell ageing to provide a kind of new strategy, is beneficial to MSCs in clinic The application of aspect.
Accompanying drawing explanation
Fig. 1 is that various concentration H of ECM effect is analyzed in SA-β-Gal dyeing detection2O2SA-β-Gal dyeing sun in stem cell after process The change of property rate；
Fig. 2 is that Flow cytometry analyzes ECM effect H2O2The change of ROS level in the early ageing stem cell of induction；
Fig. 3 is that Flow cytometry analyzes ECM effect H2O2The change of the early ageing stem cell inner cell period profile of induction；
Fig. 4 is that various concentration H of ECM effect is analyzed in RT-PCR detection2O2Intracellular p16 after processINK4αGene expression dose changes；
Fig. 5 is that intracellular p16 after ECM effect early ageing stem cell is analyzed in Western bort detectionINK4αProtein content changes.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further explained, but specific embodiment is not to the present invention It is limited in any way.Unless stated otherwise, involved in embodiment reagent, method are reagent commonly used in the art and method.
The preparation of the extracellular matrix biomaterial (ECM) of embodiment 1 anti-cell early ageing
People source UC-MSCs umbilical cord-derived mesenchymal stem cells, UC-MSCs) 37 ℃、5%CO2Incubator is incubated at 175cm2In the blake bottle of complete medium, (volume ratio of each component of culture medium is: 89% α-MEM, the hyclone of 10%, the penicillin of 1%, 1% streptomysin), within every three days, change a not good liquor.Use when cell density is close to 90% 0.25% trypsin-EDTA vitellophag, by 5000/cm2Being inoculated in orifice plate cultivation, until reaching 90% fusion, adding 100 μM ascorbic acid continues to cultivate 8 days, within three days, changes liquid once.In order to obtain free ECM after 8 days, cultivate cell and add de-cell Mixed liquor (by the phosphate buffered saline of pH7.4, containing 0.5%Triton X-100 and 20mM ammoniacal liquor) is under 37 DEG C of environment Hatch 5 minutes.The most cell free ECM PBS cleans 3 times, for the last time with the PBS dual anti-containing 1X and be retained in In orifice plate, 4 DEG C are stored in gnotobasis standby.
Embodiment 2 ECM is to various concentration H2O2SA-β-Gal stained positive rate in stem cell after process, intracellular ROS, thin The impact of born of the same parents' period profile
People source UC-MSCs is at 37 DEG C, 5%CO2Incubator is incubated at 175cm2(the volume ratio of each component of culture medium in blake bottle It is the α-MEM of 89%, the hyclone of 10%, the penicillin of 1%, streptomysin), within every three days, change a not good liquor.When cell density is close With 0.25% trypsin-EDTA vitellophag when 90%, by 5000/cm2(method is that TCPS plate is bright with 0.2% to be inoculated in pretreatment Glue is hatched 1 hour under 37 DEG C of environment, then at room temperature hatches 30 minutes with 1% glutaraldehyde and 1M monoethanolamine) plurality of specifications Orifice plate in (12 orifice plates for SA-β-Gal dye, 6 orifice plates for ROS content, the cell cycle, RT-PCR measure, 75cm2Training Support bottle for p16INK4αAnalysis of protein) cultivate, until reach 90% fusion, adds 100 μMs of ascorbic acid and continue cultivations 8 days, three It changes liquid once.In order to obtain free ECM after 8 days, cultivate the de-cell mixture of cell addition and (delayed by the phosphate of pH7.4 Rush liquid preparation, containing 0.5%Triton X-100 and 20mM ammoniacal liquor) hatch under 37 DEG C of environment 5 minutes.The most cell free ECM With PBS clean 3 times, for the last time with the PBS dual anti-containing 1X and reservation in the orifice plate, 4 DEG C are stored in gnotobasis Standby.UC-MSCs is with 5000/cm2Density is seeded in TCPS and ECM plate respectively in 37 DEG C, 5%CO2Cultivate under environment.
A. ECM is to various concentration H2O2The impact of SA-β-Gal stained positive rate in stem cell after process
SA-β-gal stained positive is always as a typical biomarker crossing presenility, and SA-β-gal dyes use SA-β-gal staining kit (green skies biological study institute).The operating instruction provided according to reagent business, cell pellet overnight is incubated in Without CO2 37 DEG C of shaking tables in, second day sucking-off dyeing liquor, after PBS twice, DAPI redyes 3 min, then with PBS 3 times, Take pictures under Olympus IX51 inverted microscope as early as possible.In order to quantify SA-'s β-gal staining positive cells (cell dyes blueness) Percentage, often group randomly chooses 10 captured pictures, and every pictures at least contains 200 cells, statistics senile cell Ratio.
Result is as it is shown in figure 1, ECM substantially reduces various concentration H2O2SA-β-gal the stained positive of UC-MSCs after process Rate.(Scale bar=100 μm)
B. ECM is to H2O2The impact of ROS changes of contents in the early ageing stem cell of induction
DCF-DA fluorescent quantitation detection intracellular ROS level.Often manage and at least collect 2 × 105Individual cell is at 10 μM 2 ', 7 ' two 37 C lucifuge water-bath 30 minutes in chlorine fluorescein oxalic acid (DCF-DA).Cytomics FC500 stream type cell analyzer is used to survey Amount fluorescence intensity.
Result is as in figure 2 it is shown, ECM significantly reduces H2O2ROS content in the early ageing UC-MSCs of induction.
C. ECM is to H2O2The impact of the early ageing stem cell inner cell period profile of induction
Cell cycle distribution is assessed by PI staining analysis.Attached cell is separated by trypsase, and the cell of separation is collected in EP Guan Zhong, 4 DEG C are fixed on 70% ethanol 24 hours.After second day washed once with the PBS of precooling, the often pipe sample PI of 100 μ l Dye liquor (configuring containing 50ug/ml PI and 50ug/ml RNase A, PBS) is at 37 DEG C
In water bath, lucifuge is hatched 30 minutes, goes up machine testing after transferring to streaming pipe as early as possible, and each sample at least detects 5000 Cell.
Result is as it is shown on figure 3, ECM adds H2O2The early ageing UC-MSCs of induction enters the ratio of S phase.
Embodiment 3 ECM is to various concentration H2O2P16 in stem cell after processINK4αGene and the impact of protein expression.
A.ECM is to various concentration H2O2P16 in stem cell after processINK4αThe ECM that affects of gene level change builds and thin Born of the same parents' training method is with embodiment 1, and attached cell is used the cracking of TRIzol reagent and then extracts total serum IgE, tries according to cDNA reverse transcription The operating procedure of agent box (Thermo Fisher company of the U.S.) takes 1ug reverse transcription from total serum IgE and becomes cDNA.In order to quantify mRNA Expression, take be equal to 50ngRNA amount cDNA use the super mixed kit (iTap of Universal fluorescence dyestuffTM Universal SYBR Green Supermix kit) at CFX96TMThe operating procedure provided according to producer on real-time PCR reactions machine is polymerized Enzyme chain reaction.We have detected SAG p16INK4α, GAPDH is as reference gene.Relevant transcriptional level is according to such as Lower formula calculates:
χ=2-△△Ct, △ △ Ct=△ E-△ C, △ E=Ctexp-CtGAPDH, △ C=Ctct1-CtGAPDH。
As shown in Figure 4, ECM significantly reduces various concentration H to result2O2P16 in UC-MSCs after processINK4αGene water Flat.
B. ECM is to H2O2P16 in the early ageing stem cell of inductionINK4αThe impact of protein content change
75cm2Transfer in 1.5ml EP pipe after the cell trypsinization that blake bottle is cultivated, after high speed centrifugation, remove supernatant, The cell pyrolysis liquid 100 μ l that often pipe adds containing protease inhibitors cracks 30 minutes on ice, takes supernatant, make after high speed centrifugation Protein concentration is measured with BCA protein quantification kit (the green skies).The albumen that equivalent is extracted becomes in 15% polyacrylamide gel Property and separation, then by electrophoretic transfer to nitrocellulose filter, the p16 that film diluted at PBSINK4α, in α-tubulin antibody 4 DEG C of night incubation, then film continues to hatch 1 hour in the horseradish peroxidase of dilution, after adding chemical luminescence reagent kit Exposure imaging as early as possible, uses ImageJ software to quantify target protein.
Result is as it is shown in figure 5, ECM considerably reduces H2O2P16 in the early ageing UC-MSCs of inductionINK4αProtein level.
To sum up experiment shows, ECM can substantially weaken H2O2SA-β-Gal activity in the early ageing UC-MSCs of induction, reduces UC- ROS content in MSCs, increases cell and enters S phase ratio, reduce p16 in UC-MSCsINK4αGene and protein level, effectively alleviate H2O2The UC-MSCs early ageing of induction, in the field such as cellular transplantation therapy and regenerative medicine, the prospect of being widely applied.

Claims (10)

1. the extracellular matrix biomaterial of an anti-cell early ageing, it is characterised in that be to cultivate umbilical cord mesenchymal stem cells to divide The matrix secreted, can the early ageing of anti-cell.
2. the preparation method of the extracellular matrix biomaterial of the anti-cell early ageing described in claim 1, it is characterised in that include Following steps:
1) umbilical cord mesenchymal stem cells is cultivated in complete medium, when cell density is close to 90%, add containing Vitamin C The complete culture solution of acid is cultivated 7-10 days；
2) add de-cell liquid, build and obtain extracellular matrix biomaterial.
The preparation method of the extracellular matrix biomaterial of anti-cell early ageing the most according to claim 2, it is characterised in that Described umbilical cord mesenchymal stem cells takes from people source, pig source, mouse source or rabbit source.
The preparation method of the extracellular matrix biomaterial of anti-cell early ageing the most according to claim 2, it is characterised in that Described complete medium be component be α-MEM 80 ~ 90% volume ratio, hyclone 10 ~ 20% volume ratio, antibiotic 10 ~ 200 U/mL。
The preparation method of the extracellular matrix biomaterial of anti-cell early ageing the most according to claim 2, it is characterised in that In described complete culture solution, the concentration of ascorbic acid is 100-200 μM.
The preparation method of the extracellular matrix biomaterial of anti-cell early ageing the most according to claim 2, it is characterised in that Described de-cell liquid is formed by the phosphate buffered saline of pH7.4, containing Triton X-100 0.5 ~ 5% volume ratio and ammonia Water 10-40mM.
7. the extracellular matrix biomaterial of anti-cell early ageing described in claim 1 is at cell transplantation, regenerative medicine or tissue Application in engineering.
8. the application in cell is cultivated of the extracellular matrix biomaterial of anti-cell early ageing described in claim 1.
Application the most according to claim 8, it is characterised in that cell to be cultivated is seeded in the thin of described anti-cell early ageing In extracellular matrix biomaterial culture plate, at 37 DEG C, 5% CO2Cultivate under environment.
10. the extracellular matrix biomaterial of anti-cell early ageing described in claim 1 is preparing the old and feeble cosmetics of anti-cell, medicine Application in product or health products.
CN201610202228.8A 2016-04-01 2016-04-01 Extracellular matrix biomaterial for resisting cell premature senility and preparation method and application thereof Pending CN105854084A (en)

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