CN106282101A - A kind of promote the human amnion mesenchymal stem cell method to Chondrocyte Differentiation and application - Google Patents
A kind of promote the human amnion mesenchymal stem cell method to Chondrocyte Differentiation and application Download PDFInfo
- Publication number
- CN106282101A CN106282101A CN201610941208.2A CN201610941208A CN106282101A CN 106282101 A CN106282101 A CN 106282101A CN 201610941208 A CN201610941208 A CN 201610941208A CN 106282101 A CN106282101 A CN 106282101A
- Authority
- CN
- China
- Prior art keywords
- mesenchymal stem
- stem cell
- cell
- differentiation
- human amnion
- Prior art date
- 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
Links
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
- C12N5/0655—Chondrocytes; Cartilage
-
- 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
- A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
-
- 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
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/90—Polysaccharides
- C12N2501/905—Hyaluronic acid
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1392—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from mesenchymal stem cells from other natural sources
-
- 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
Abstract
The present invention relates to a kind of promote the human amnion mesenchymal stem cell method to Chondrocyte Differentiation, the method is interpolation inducing composition in human amnion mesenchymal stem cell, and described inducing composition includes 0.01～2g/L hyaluronic acid and 0～100mg/L ascorbic acid.The present invention is compared with conventional abductive approach, it is remarkably improved human amnion mesenchymal stem cell to Chondrocyte Differentiation, glycosaminoglycans and II collagen type are expressed in strong positive, the expression of the Subchondral drilling related genes such as Col2 α 1, ACAN and Sox9 and albumen is the most substantially raised, and can significantly improve the human amnion mesenchymal stem cell efficiency to Chondrocyte Differentiation.In the present invention, inducing composition can be used for human mesenchymal stem cell in Chondrocyte Differentiation and in cartilage injury's disease.
Description
Technical field
The invention belongs to stem cell biological technology and bone tissue engineer technical field, be related specifically to a kind of promotion people's amniotic membrane
The method of mesenchymal stem cells into chondrocytes differentiation and application.
Background technology
The joint caused cartilage injurys of reason such as athletic injury, degeneration, infection, tumor resection, are in orthopaedic disease
Commonly encountered diseases, frequently-occurring disease, and become the important diseases affecting human health.Owing to articular cartilage does not has blood supply, and chondrocyte bag
It is embedded in the extracellular matrix of stiff, it is difficult to move to damaged part and participate in repairing.But, traditional therapy, curative effect extremely has
Limit.Articular cartilage defect is always Orthopedic Clinical disease treatment difficult point and the important topic of research.At present, treat cartilage injury's
Means mainly include that autologous or homogenous cartilage piece of tissue is transplanted, Autologous Chondrocyte is transplanted, implant biological support etc..These treatments
Although cartilage injury is repaired by method a certain effect, but there is donor limited source, graft is difficult to make up with health cartilage,
Easily cause joint fibrosis complication, the problems such as repairing effect is the best.Cartilage tissue engineered is to solve this difficult problem to provide newly
Thinking.Utilize organizational project means to build tissue engineering bone/cartilage and can greatly reduce above many frauds for treating cartilage injury
End, brings new hope for solving this difficult problem.But, cartilage tissue engineered seed cell urgently to be resolved hurrily, growth regulator
With three big bottleneck problems such as timbering materials.
Seed cell is the first element building organizational project, is the premise carrying out Tissue Engineering Study and basis.In a large number
Research show, owing to human amnion mesenchymal stem cell has the amplification ability more higher than mesenchymal stem cells MSCs, differentiation potency
Power, reduced immunogenicity, without advantages such as tumorigenesis risk and ethics restrictions it is considered to be the preferable seed cell of regenerative medicine, also
Be cartilage tissue engineered preferable seed cell source (Xiao Jianhui. people amniotic membrane stem cell: the ideal kind of regenerative medicine is careful
Born of the same parents' resource. Zunyi Medical College journal 2015,38:439-449).The early-stage Study prompting of this laboratory, uses existing bone marrow
The one-tenth cartilage differentiation growth regulating inducible factor of mescenchymal stem cell, the chondroblast that human amnion mesenchymal stem cell shows
The ability of differentiation.But, other mescenchymal stem cell similar becomes cartilage differentiation to report result, existing induction differentiated system, needs
Special, expensive growth regulator, such as basic fibroblast growth factor (bFGF), transforming growth factor β
(TGF-β), bone morphogenetic protein (BMP), type-1 insulin like growth factor (IGF-1), hepatocyte growth factor (HGF) and blood
Platelet source somatomedin (PDGF) etc., and these growth regulators are vivo biodistribution macromole, not only get involved in internal in length and breadth
Staggered physiological process, and in extremely complex multiple adjusting function.This is also that current stem cell induces into cartilage differentiation effect
The low main cause of rate.In recent years, also have been reported that poisonous snake nerve growth factor, melatonin etc. can induce stem cell to become cartilage differentiation
(Zheng Li, thunder painting, Lu Zhenhui, etc. the method that Recombinant Naja naja atra NGF induction stem cell becomes cartilage differentiation. Shen
Please numbers 201410373621.4；Gong Yihong, He Fan, Liu Xiaozhen, etc. in preparation, melatonin promotes that mescenchymal stem cell becomes soft
Application in bone differentiation medicament. application number 201310383987.5), but, there is also induction stem cell equally and become cartilage differentiation
The problems such as efficiency is low.And, as exogenous factor, there is the risk being identified by human immune system and attacking.It would therefore be highly desirable to
Find more efficient, safe, economic growth regulator or induction differentiated system, promote the development of cartilage tissue engineered technology,
Service extensive patients.
Hyaluronic acid is human body cell epimatrix principal structural component.And extracellular matrix determines the shape of cell, impact
The vigor of cell, migration, proliferation and differentiation, and participate in signal transmission, hyaluronic acid plays key player wherein, and at body
Performance multi-biological function under normal and pathological state.Hyaluronic acid be a kind of byD-glucuronic acid,N-acetylaminohydroxyphenylarsonic acidD-glucose, with-1, the non-protein class straight chain polymer acid mucopolysaccharide that 3 glycosidic bonds and the handing-over of-Isosorbide-5-Nitrae glycosidic bond are formed is transparent
The biological function of matter acid has close relationship with himself molecular size range, dosage, cell density etc..Such as, Kumta research
Group reports 60kDa, the hyaluronic acid of 900 kDa and the 2100kDa molecular weight proliferation and differentiation to children's Mus braincap mesenchymal cell
Effect.Result is pointed out, and the HA of low-molecular-weight can promote cell proliferation, and be enhanced to bone cell differentiation specific marker in dose-dependant
The expression of Bone Gla protein gene mRNA, but to the index such as alkali phosphatase, bone formation without significant change, and other two molecular weight
HA all can be obviously promoted osteoprogenitor cells propagation and induce differentiation into osteocyte, and Kimata etc. observes HA and regulates exponential phase Mus table
Skin fibroblast proliferation situation, finds to there is positive and negative effect regulation phenomenon, and this depends entirely on addition range of doses
Cell density during HA, relative high density is cultivated and is promoted propagation trend after dosing in dose-dependant, on the contrary Inhibit proliferaton.Therefore,
Research worker is first with human amnion mesenchymal stem cell as seed cell, and screening specified molecular weight hyaluronic acid is Main Factors
Inducing composition, and set up and induce differentiated system accordingly, efficiently promote that human amnion mesenchymal stem cell breaks up to chondroblast,
It is used for treating cartilage injury for structure tissue engineering bone/cartilage to lay the foundation.
Summary of the invention
For the deficiencies in the prior art, it is an object of the invention to provide a kind of safe efficient, economic promotion people's amniotic membrane
The method that mescenchymal stem cell breaks up to chondroblast.
A kind of promoting the human amnion mesenchymal stem cell method to Chondrocyte Differentiation, it concretely comprises the following steps:
(1) separation and Culture of human amnion mesenchymal stem cell: health full term Cesarean esction fresh human placenta passivity mechanical stripping amniotic membrane, uses
Amniotic membrane is repeatedly rinsed, after removing residual bloodstain and surface mucus containing 1% dual anti-D-Hank ' s liquid；Amniotic membrane is cut into 1～3 cm2
Size, is sub-packed in 50mL centrifuge tube, adds the 0.05% pancreas egg containing 0.02% EDTA-2Na of about 2 times of volumes of amnion tissue
White enzymic digestion liquid, in 35～37 DEG C of constant water bath box, 150～185rpm rotate digestion about 30 min, filter through 300 mesh rustless steels
After net filtration, discard the Digestive system containing amniotic epithelial cells, rejoin fresh containing the 0.05% of 0.02% EDTA-2Na
Tryptic digestive juice, repeats to digest once, again after 300 mesh stainless steel filtering nets filter, discards Digestive system；Through above-mentioned pancreas egg
After white enzymic digestion, remaining amnion tissue cleans once with D-Hank ' the s liquid dual anti-containing 1%, and the trypsin removing residual disappears
Change liquid, add the 0.5 mg/mL II Collagenase Type Digestive system containing 0.05 mg/mL DNase I, in 35～37 DEG C of waters bath with thermostatic control
In case, 150～185 rpm rotate digestion 1～2 h, until remaining amnion tissue fragment catapepsis becomes cotton-shaped, through 300 mesh
Stainless (steel) wire filters, and collects cell filtrate, 1500rpm, 4 DEG C of centrifugal 10 min, abandons supernatant, and cell precipitation is i.e. for filling between people's amniotic membrane
Matter stem cell；With the low sugar DMEM re-suspended cell containing 10% hyclone, plant in T25 Tissue Culture Flask, at 35～37 DEG C, containing 1～
10%CO2And 85～100% saturation of the air humidity constant temperature culture, treat that cell degrees of fusion reaches 80% Secondary Culture carried out above；
(2) the induction human amnion mesenchymal stem cell preparation to chondroblast induction compositions: complete at 1L DMEM in high glucose
In full culture medium, add containing 0.01～2 g/L and the inducing composition of 0～100mg/L ascorbic acid, obtain human amnion mesenchymal
Stem cell is to the inducing composition of Chondrocyte Differentiation；
(3) inducing composition promotees the human amnion mesenchymal stem cell method to Chondrocyte Differentiation: take 2 generation exponential phase people sheep
Intermembranous mesenchymal stem cells, by 2 × 105The cell concentration in cells/ hole, is inoculated in 6 orifice plates, adds induction group after 0～48 times
Compound, changes once application substrate and inducing composition for every 2～3 days, 35～37 DEG C, containing 1～10% CO2And 85～100% air satisfy
Cultivating 14～28 days with in humidity constant incubator, induction differentiation terminates.
For inducing the human amnion mesenchymal stem cell of differentiation to be 2～5 generations.
For inducing the human amnion mesenchymal stem cell initial seed density of differentiation, by 5 × 105～5 × 106Individual/bottle
Cell-seeding-density kind is in 25cm2Culture bottle.
In inducing composition, the molecular weight of hyaluronic acid is 20～2200KD.
Scheme of the present invention regulation and control human amnion mesenchymal stem cell breaks up to chondroblast, can be thin as cartilage injury
Born of the same parents transplant and the purposes of cartilage tissue engineered seed cell.
Scheme of the present invention regulation and control human amnion mesenchymal stem cell breaks up to chondroblast, can replace as seed cell
For chondroblast, it is to avoid high immunogenicity problem after external acquisition chondrocyte difficulty and chondrocyte cell transplantation.
Inducing composition of the present invention can prepare treatment cartilage injury's medicine, and associating human amnion mesenchymal stem cell is transplanted
The relevant cartilage injury's disease for the treatment of.
Accompanying drawing explanation
Fig. 1 is the morphological characteristic figure (× 100) after human amnion mesenchymal stem cell induction is broken up 28 days, (a) matched group,
B () positive controls, (c) adds the medicine group of inducing composition；
Fig. 2 be Toluidine blue staining detection human amnion mesenchymal stem cell induction differentiation 28 days after glycosaminoglycans secretion situation (×
200), (a) matched group, (b) positive controls, (c) adds the medicine group of inducing composition；
Fig. 3 is the expression becoming cartilage related gene after human amnion mesenchymal stem cell induction is broken up 28 days；
Fig. 4 is the expression of dryness gene after human amnion mesenchymal stem cell induction is broken up 28 days；
Fig. 5 is the expression of the skeletonization associated protein after human amnion mesenchymal stem cell induction is broken up 28 days.
Detailed description of the invention
The present invention is described further by below embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.
Embodiment 1: promote the human amnion mesenchymal stem cell method to Chondrocyte Differentiation efficiency and application
1, the separation and Culture of human amnion mesenchymal stem cell
Health full term Cesarean esction fresh human placenta passivity mechanical stripping amniotic membrane, with D-Hank ' s liquid (the final concentration of green grass or young crops dual anti-containing 1%
Mycin: 100 IU/mL, streptomycin: 100 IU/mL, before use Fresh) repeatedly rinse amniotic membrane, remove residual bloodstain and table
After the mucus of face.Amniotic membrane is cut into 1～3 cm2Size, is sub-packed in 50mL centrifuge tube, adds containing of about 2 times of volumes of amnion tissue
0.05% tryptic digestive juice of 0.02% EDTA-2Na, in 35～37 DEG C of constant water bath box, 150～185rpm rotate
Digestion about 30 min, after 300 mesh stainless steel filtering nets filter, discard the Digestive system containing amniotic epithelial cells, rejoin new
Fresh 0.05% tryptic digestive juice containing 0.02% EDTA-2Na, repeats to digest once, again filters through 300 mesh rustless steels
After net filtration, discard Digestive system.After above-mentioned trypsinization, remaining amnion tissue is with D-Hank ' the s liquid dual anti-containing 1%
Clean once, remove the tryptic digestive juice of residual, add the 0.5 mg/mL II type glue containing 0.05 mg/mL DNase I
Protoenzyme Digestive system, in 35～37 DEG C of constant water bath box, 150～185rpm rotate digestion 1～2 h, until remaining amniotic membrane group
Knit fragment catapepsis and become cotton-shaped, filter through 300 mesh stainless (steel) wires, collection cell filtrate (i.e. Digestive system), 1500 rpm, 4 DEG C
Centrifugal 10 min, abandon supernatant, and cell precipitation is human amnion mesenchymal stem cell.With the low sugar DMEM weight containing 10% hyclone
Outstanding cell, plants in T25 Tissue Culture Flask, at 35～37 DEG C, containing 1～10% CO2And 85～100% the saturation of the air humidity constant temperature training
Supporting, treat that cell degrees of fusion reaches 80% Secondary Culture carried out above, the 2nd～5 is alternative in following experiment.
2, the induction human amnion mesenchymal stem cell preparation to chondroblast induction compositions
In 1L DMEM in high glucose complete medium, add containing 0.01～2 g/L, 20～2200KD hyaluronic acids and 0～100mg/
The inducing composition of L ascorbic acid, i.e. obtains and improves the human amnion mesenchymal stem cell induction group to Chondrocyte Differentiation efficiency
Compound.
3, inducing composition promotees the human amnion mesenchymal stem cell application to Chondrocyte Differentiation
Take 2 generation exponential phase human amnion mesenchymal stem cells, by 2 × 105The cell concentration in cells/ hole, is inoculated in 6 orifice plates
In, add inducing composition after 0～48 hour, within every 2 days, change once application substrate and inducing composition.Cultivate 7～28 days continuously.
Cell-based assay is tested: observation of cell metamorphosis under optical microscope；Reflection chondroblast level of differentiation
Index includes that glycosaminoglycans secretion and II collagen type are expressed.Use toluidine blue dyeing to measure glycosaminoglycans, use immunity
Cytochemistry measures II collagen type and expresses.
When cultivating by 28 days, compared with matched group, positive controls (positive group) and add the medicine group of inducing composition
The cell that (medicine group) induces is become paving stone shape by spindle shape, and matched group majority cell is still in spindle shape (Fig. 1)；In training
When supporting 28 days, toluidine blue dyes, and compared with matched group, positive group and experimental group all have the positive cell in deep blue purple color,
And medicine group positive cell color is deeper (Fig. 2) than positive group, prompting inducing composition has stronger one-tenth cartilage ability.
Gene expression testing result: use RT-qPCR standard measure to determine Col2 α 1, ACAN with Sox9 etc. and become cartilage thin
The expression of born of the same parents' related gene, usesβ-actin is as internal reference.When cultivating by 28 days, compared with matched group, positive control
Organizing and become cartilage related gene expression substantially to raise in medicine group, prompting inducing composition has stronger one-tenth cartilage ability.And medicine
Thing group is again apparently higher than positive group (Fig. 3).And, the expression of dryness gene Nanog, Oct4 gene of medicine group is the brightest
Aobvious enhancing (Fig. 4).Therefore, inducing composition is conducive to maintaining the differentiation capability of human amnion mesenchymal stem cell.
Protein expression testing result: use Western blotting method to have detected Col2 α 1, ACAN with Sox9 etc. and become soft
The expression of osteocyte associated protein, withβ-actin is as internal reference.When cultivating by 28 days, compared with matched group, positive
Matched group with medicine group becomes cartilage correlative protein expression substantially raise (Fig. 5), point out it to Chondrocyte Differentiation.
Embodiment 2. promotes that human amnion mesenchymal stem cell chondroblast is in the purposes of other side
Human amnion mesenchymal stem cell breaks up to chondroblast with inducing composition of the present invention induction in vitro, can be as cartilage
The purposes of the seed cell of organizational project and cell transplantation.Cartilage injury's disease all there is certain preventive and therapeutic effect.Use the present invention
Inducing composition inducing differentiation of human amnion mesenchymal stem cell substitutes chondrocyte as seed cell, can avoid chondrocyte body
Outer more difficult acquisition and the problem of high immunogenicity.
Claims (5)
1. one kind promotes the human amnion mesenchymal stem cell method to Chondrocyte Differentiation, it is characterised in that: this promotion people's amniotic membrane
Concretely comprising the following steps of mesenchymal stem cells into chondrocytes differentiation:
(1) separation and Culture of human amnion mesenchymal stem cell: by health full term Cesarean esction fresh human placenta passivity mechanical stripping amniotic membrane,
Amniotic membrane is repeatedly rinsed, after removing residual bloodstain and surface mucus with D-Hank ' the s liquid dual anti-containing 1%；Amniotic membrane is cut into 1～3
cm2Size, is sub-packed in 50mL centrifuge tube, adds 0.05% pancreas containing 0.02% EDTA-2Na of about 2 times of volumes of amnion tissue
Protease digestion liquid, in 35～37 DEG C of constant water bath box, 150～185rpm rotate digestion about 30 min, through 300 mesh rustless steels
After strainer filtering, discard the Digestive system containing amniotic epithelial cells, rejoin fresh containing 0.02% EDTA-2Na's
0.05% tryptic digestive juice, repeats to digest once, again after 300 mesh stainless steel filtering nets filter, discards Digestive system；Through upper
After stating trypsinization, remaining amnion tissue cleans once with D-Hank ' the s liquid dual anti-containing 1%, removes the pancreas egg of residual
White enzymic digestion liquid, adds the 0.5 mg/mL II Collagenase Type Digestive system containing 0.05 mg/mL DNase I, in 35～37 DEG C of perseverances
In warm water bath cabinet, 150～185 rpm rotate digestion 1～2 h, until remaining amnion tissue fragment catapepsis becomes cotton-shaped, warp
300 mesh stainless (steel) wires filter, and collect cell filtrate, 1500rpm, 4 DEG C of centrifugal 10 min, abandon supernatant, and cell precipitation is i.e. people sheep
Intermembranous mesenchymal stem cells；With the low sugar DMEM re-suspended cell containing 10% hyclone, plant in T25 Tissue Culture Flask, 35～37
DEG C, containing 1～10%CO2And 85～100% saturation of the air humidity constant temperature culture, treat that cell degrees of fusion reaches 80% and carried out above passes on training
Support；
(2) the induction human amnion mesenchymal stem cell preparation to chondroblast induction compositions: complete at 1L DMEM in high glucose
In full culture medium, add containing 0.01～2 g/L hyaluronic acids and the inducing composition of 0～100mg/L ascorbic acid, obtain people sheep
Intermembranous mesenchymal stem cells is to the inducing composition of Chondrocyte Differentiation；
(3) inducing composition promotes the human amnion mesenchymal stem cell method to Chondrocyte Differentiation: take 2 generation exponential phase people
Amnion mesenchymal stem cell, by 2 × 105 The cell concentration in cells/ hole, is inoculated in 6 orifice plates, adds induction group after 0～48 times
Compound, changes once application substrate and inducing composition for every 2～3 days, 35～37 DEG C, containing 1～10% CO2And 85～100% air satisfy
Cultivating 14～28 days with in humidity constant incubator, induction differentiation terminates.
Promote the human amnion mesenchymal stem cell method to Chondrocyte Differentiation the most according to claim 1, it is characterised in that:
For inducing the human amnion mesenchymal stem cell of differentiation to be 2～5 generations.
Promote the human amnion mesenchymal stem cell method to Chondrocyte Differentiation the most according to claim 1, it is characterised in that:
For inducing the human amnion mesenchymal stem cell initial seed density of differentiation, by 5 × 105～5 × 106The cell inoculation of individual/bottle is close
Degree kind is in 25cm2Culture bottle.
Promote the human amnion mesenchymal stem cell method to Chondrocyte Differentiation the most according to claim 1, it is characterised in that:
In inducing composition, the molecular weight of hyaluronic acid is 20～2200KD.
Promote that human amnion mesenchymal stem cell can be applicable to cartilage injury to chondroblast differentiation the most according to claim 1
Cell transplantation and cartilage tissue engineered seed cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610941208.2A CN106282101A (en) | 2016-11-02 | 2016-11-02 | A kind of promote the human amnion mesenchymal stem cell method to Chondrocyte Differentiation and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610941208.2A CN106282101A (en) | 2016-11-02 | 2016-11-02 | A kind of promote the human amnion mesenchymal stem cell method to Chondrocyte Differentiation and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106282101A true CN106282101A (en) | 2017-01-04 |
Family
ID=57719488
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610941208.2A Pending CN106282101A (en) | 2016-11-02 | 2016-11-02 | A kind of promote the human amnion mesenchymal stem cell method to Chondrocyte Differentiation and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106282101A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103010A (en) * | 2018-02-06 | 2018-06-01 | 宁波东钱湖旅游度假区靖芮医疗美容诊所有限公司 | A kind of method of amnion stem cell extraction, culture and purifying |
| CN113384753A (en) * | 2021-03-19 | 2021-09-14 | 杭州协合医疗用品有限公司 | Injectable temperature-sensitive composite hydrogel containing adipose-derived mesenchymal stem cells and preparation method and application thereof |
| CN115820546A (en) * | 2022-04-12 | 2023-03-21 | 中国人民解放军军事科学院军事医学研究院 | Method for promoting chondrogenic differentiation of brown adipose-derived stem cells and application of method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914490A (en) * | 2010-08-13 | 2010-12-15 | 中国医科大学 | Human amnion mesenchymal stem cell serum-free culture medium and culture method thereof |
| CN102191218A (en) * | 2011-03-28 | 2011-09-21 | 遵义医学院附属医院 | Complete medium and human amnion-derived mesenchymal stem cell culture method |
| CN102559586A (en) * | 2011-12-08 | 2012-07-11 | 遵义医学院附属医院 | Separation, purification and identification methods of human amnion mesenchymal stem cells |
| CN105695402A (en) * | 2016-04-14 | 2016-06-22 | 安沂华 | Composition and method for inducing mesenchymal stem cells to be differentiated to cartilage cells |
| CN106039408A (en) * | 2015-04-07 | 2016-10-26 | 四川蓝光英诺生物科技股份有限公司 | Preparation method for biobricks containing mesenchymal stem cell (MSC) and biobrick prepared by using same |
-
2016
- 2016-11-02 CN CN201610941208.2A patent/CN106282101A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914490A (en) * | 2010-08-13 | 2010-12-15 | 中国医科大学 | Human amnion mesenchymal stem cell serum-free culture medium and culture method thereof |
| CN102191218A (en) * | 2011-03-28 | 2011-09-21 | 遵义医学院附属医院 | Complete medium and human amnion-derived mesenchymal stem cell culture method |
| CN102559586A (en) * | 2011-12-08 | 2012-07-11 | 遵义医学院附属医院 | Separation, purification and identification methods of human amnion mesenchymal stem cells |
| CN106039408A (en) * | 2015-04-07 | 2016-10-26 | 四川蓝光英诺生物科技股份有限公司 | Preparation method for biobricks containing mesenchymal stem cell (MSC) and biobrick prepared by using same |
| CN105695402A (en) * | 2016-04-14 | 2016-06-22 | 安沂华 | Composition and method for inducing mesenchymal stem cells to be differentiated to cartilage cells |
Non-Patent Citations (2)
| Title |
|---|
| 宋秀军等: "人羊膜间充质细胞具有分化成软骨及成骨细胞的潜能", 《中国组织工程研究与临床康复》 * |
| 张清芳等: "透明质酸在间充质干细胞向软骨细胞分化中的应用", 《中国生物工程杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103010A (en) * | 2018-02-06 | 2018-06-01 | 宁波东钱湖旅游度假区靖芮医疗美容诊所有限公司 | A kind of method of amnion stem cell extraction, culture and purifying |
| CN113384753A (en) * | 2021-03-19 | 2021-09-14 | 杭州协合医疗用品有限公司 | Injectable temperature-sensitive composite hydrogel containing adipose-derived mesenchymal stem cells and preparation method and application thereof |
| CN115820546A (en) * | 2022-04-12 | 2023-03-21 | 中国人民解放军军事科学院军事医学研究院 | Method for promoting chondrogenic differentiation of brown adipose-derived stem cells and application of method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106109496B (en) | Human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation method | |
| CN108823156A (en) | For the clinical grade human umbilical cord mesenchymal stem cells composite factor of reparation and the preparation method of freeze-dried powder | |
| CN104263697B (en) | A kind of method that inducing culture and induction human adipose mesenchymal stem cells generate insulin secretory cell | |
| CN106754674B (en) | The method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion | |
| CN108309822A (en) | A kind of preparation method of human umbilical cord mesenchymal stem cells paracrine factor freeze-dried powder | |
| CN104726406B (en) | It is a kind of to induce the method that dental pulp Derived from Mesenchymal Stem Cells is nerve cell | |
| CN104321426A (en) | Immortalized stem cells and medicinal composition and medicinal preparation comprising product thereof as active ingredient | |
| CN101757691A (en) | Preparation method of tissue engineering cornea | |
| CN108192862A (en) | A kind of preparation method of pilose antler stem cell, pilose antler stem cell and its application | |
| CN103480040A (en) | Bone matrix material containing various proteins secreted by umbilical cord mesenchymal stem cells and preparation method thereof | |
| CN108057116A (en) | Application of the stem cell composition in skin injury medicine | |
| CN107267451A (en) | A kind of preparation method of primary dental pulp stem cell and the method for building dental pulp stem cell storehouse | |
| CN105154407A (en) | Preparation method and application of human adipose-derived stem cells for improving skin repairing function | |
| CN105013014A (en) | Preparation method and application of acellular matrix biological material | |
| CN108865986A (en) | For repairing articular cartilage damage/defect mescenchymal stem cell preparation and its preparation method and application | |
| CN104651305A (en) | Method for acquiring bioactive proteins by utilizing umbilical cord mesenchymal stem cells | |
| CN103223194A (en) | Cartilage graft for cartilage injury repair and preparation method thereof | |
| CN110507597A (en) | A kind of composition and preparation method thereof, application | |
| CN106282101A (en) | A kind of promote the human amnion mesenchymal stem cell method to Chondrocyte Differentiation and application | |
| CN105106240B (en) | A kind of stem cell medicine and its purposes in vascular interventional treatment cerebral apoplexy | |
| CN104250655A (en) | BMP(bone morphogenetic protein)-2/VEGF(vascular endothelial growth factor)165 double gene modified bone marrow mesenchymal stem cells and preparation method thereof | |
| CN105838672B (en) | Stem cell induction broth, preparation method and its application for eye | |
| CN114874982A (en) | Culture method for enhancing umbilical cord mesenchymal stem cells to secrete vascular endothelial growth factors | |
| CN106701670A (en) | Methods for enhancing bioactive factor secretion capacity of mesenchymal stem cells and extracting active factors in culture solution | |
| CN108066750A (en) | Stem cell and its secretion are used to treat the new application of skin burn |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170104 |