CN111690599A - Method for promoting mesenchymal stem cells to differentiate into chondroblasts - Google Patents
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Abstract
A method for promoting mesenchymal stem cells to differentiate into chondroblasts relates to the field of stem cells, in particular to a method for differentiating mesenchymal stem cells into chondroblasts. The method aims to solve the problems of small number of chondrocytes and low reproductive capacity of the existing method for differentiating the mesenchymal stem cells into the chondrocytes. The method comprises the following steps: recovering umbilical cord-derived mesenchymal stem cells from P8 generation, culturing by adopting complete culture medium containing vitamin D, vitamin A and EGF, digesting, and carrying out passage to obtain umbilical cord mesenchymal stem cells from P10 generation; taking P10 generation umbilical cord mesenchymal stem cells, cleaning, sampling for cell counting, and culturing by adopting a complete culture medium containing vitamin D, vitamin A and EGF; changing the culture medium the next day, and performing differentiation culture; using a differentiation basal medium containing vitamin D, vitamin A and TGF-beta 1; the culture is completed after 13-15 days. The invention is applied to the field of differentiation of mesenchymal stem cells into chondrocytes.
Description
Technical Field
The invention relates to the field of stem cells, in particular to a method for differentiating mesenchymal stem cells into chondroblasts.
Background
Mesenchymal stem cells are important constituent members in a stem cell family in cell biology, are derived from mesoderm and ectoderm of an embryo in an early development stage, are initially found in bone marrow of people, are mainly characterized by self-renewal, multidirectional differentiation, hematopoietic support, immune regulation and the like in clinical characteristics, and are more and more paid attention to the people in clinical use. Under certain induction conditions in vivo or in vitro, the mesenchymal stem cells can be differentiated into various human tissue cells such as fat, muscle, cardiac muscle, tendon, ligament, liver, bone, cartilage, endothelium and the like, can still have the potential of multidirectional differentiation by adopting a continuous subculture and cryopreservation method, are frequently used for repairing the injury of ideal tissues and organs in clinic, particularly nerve injury, and can also be used for treating various blood system diseases and cardiovascular diseases by adopting the mesenchymal stem cells.
Chondrocytes are induced in three stages: in the first stage, MSCs form high-density cell aggregates, stop proliferation, secrete abundant hyaluronic acid and type I collagen, and accumulate tissue-specific transcription factors such as Sox-9 and structural proteins; in the second stage, MSCs are differentiated into cartilage precursor progenitor cells, the aggregated cells are proliferated again to generate initial extracellular matrix, and cartilage differentiation is started, wherein the process is regulated and controlled by Sox-9, Sox-5, Sox-6, FGF and TGF-beta 1 signaling pathways; the third stage, called the dedifferentiation stage, is the stage where the cartilage matrix is formed, expressing collagen II, collagen IX and collagen XI.
In most experiments, the differentiation of mesenchymal stem cells into chondrocytes requires that the generation of mesenchymal stem cells is low, the number of differentiated chondrocytes is not large, and the proliferation function of cells is reduced in the process of differentiating mesenchymal stem cells of high generation into chondrocytes, resulting in the reduction of the number of differentiated chondrocytes.
Disclosure of Invention
The invention aims to solve the problems of small number of chondrocytes and low reproductive capacity of the existing method for differentiating the mesenchymal stem cells into the chondrocytes, and provides a method for promoting the differentiation of the mesenchymal stem cells into the chondroblasts.
The method for promoting the mesenchymal stem cells to be differentiated into the chondroblasts comprises the following steps:
firstly, stem cell amplification culture:
recovering umbilical cord source mesenchymal stem cells from P8 generation, culturing by adopting a serum-free culture medium, changing the culture solution the next day, wherein the culture medium used for changing the culture solution is a complete culture medium containing 0.8-1.5mol/L vitamin D, 1.5-2.5mol/L vitamin A and 90-110ng/ml EGF at the final concentration, digesting, passaging and finally obtaining the umbilical cord mesenchymal stem cells from P10 generation;
secondly, differentiation of chondroblasts:
taking the P10 substitute umbilical cord mesenchymal stem cells obtained by the culture in the step one, cleaning, sampling for cell counting, and adjusting the cell concentration to 1 × 104Per ml;
changing the culture medium the next day, namely changing the culture medium for the first time, and performing differential culture; the culture medium used for the first liquid change is a differentiation basal culture medium containing 1.8-2.2mol/L vitamin D, 3.5-4.5mol/L vitamin A and 190-210ng/ml TGF-beta 1;
then changing the culture medium every 3 days, wherein the culture medium used for changing the culture medium for the second time is a differentiation basic culture medium containing 1.8-2.2mol/L vitamin D, 3.5-4.5mol/L vitamin A and 390-410ng/ml TGF-beta 1, and the culture medium used for changing the culture medium is the same as the culture medium used for changing the culture medium for the second time;
and culturing for 13-15 days to complete the differentiation culture of the chondroblasts.
Further, in the step one, when the cell fusion degree reaches more than 70%, digesting to obtain single cells.
Further, step one is as per 1 × 106Inoculating each cell/T175 flask, and carrying out passage after the cell fusion degree reaches more than 90%.
Further, performing immune label detection on the umbilical cord mesenchymal stem cells of the generation P10 obtained in the first step.
Further, the complete medium in the first step and the second step is the basic medium added with heparin sodium, and each 560mL of the basic medium is added with 160. mu.L of heparin sodium 120-.
Further, in step two, the differentiation basal medium is MesenCultMTSodium heparin was added to the OsteogenicDifference Kit every 560mL MesenCultMT120-160. mu.l heparin sodium was added to the Osteogenic Differencention kit.
Further, in the second step, before the cells are inoculated in the 24-well plate, the slide is added, 500. mu.l of physiological saline is added, and the physiological saline is discarded after 30-35 min.
The principle of the invention is as follows:
the invention promotes the proliferation of the mesenchymal stem cells and the differentiation of the mesenchymal stem cells into bone cells by adding vitamin D, vitamin A, EGF and TGF-beta 1 into a culture medium in the processes of cell culture and differentiation.
Vitamin A has important influence on vision change, skeleton development, spermatogenesis and embryonic development, and maintains multiple physiological functions of the body. Vitamin a maintains the morphology and viability of sperm cells and also promotes the proliferation of a variety of cells. The vitamin A is more beneficial to the in-vitro proliferation of the stem cells, has a promoting effect on the aspects of the development, the meiosis and the like of the stem cells, and is also beneficial to the maintenance of the totipotency of the stem cells. Vitamin A may have a certain promotion effect on cell proliferation, and MTT detection shows that when vitamin A with a proper concentration is added into a culture solution to culture cells, the cell number is obviously increased, and the cell proliferation rate is also obviously accelerated.
Vitamin D (vitamin D, VitD) fat-soluble vitamin, which is a steroid hormone, has a wide range of physiological actions, and in addition to being widely known to participate in calcium and phosphorus metabolism in vivo, VitD also has various actions such as regulating immunity, controlling cell growth and survival, cell differentiation, protecting DNA, and the like, and also has important effects on the proliferation and differentiation of stem cells in each germ layer, particularly on the differentiation of stem cells in the nerve, osteogenic, hematopoietic, and other directions.
The Epidermal Growth Factor (EGF) is an important growth factor, is a member of an EGF-like large family, can promote the proliferation and differentiation of cells, and has strong mitogenic action on various histiocytes in vivo and in vitro.
The cartilage tissue only contains single cartilage cells, and TGF-beta 1 is added in the culture process of the MSCs, so that the proliferation of the cells and the production of extracellular matrix can be promoted, the type I collagen is reduced, and the expression of type II collagen and glycosaminoglycan (glycoaminoglycan) is up-regulated. Chondrocytes synthesize mainly extracellular matrix components such as type ii collagen and proteoglycans, and within a solid matrix, 50% -75% are collagen and 15% -30% are proteoglycans.
The invention has the beneficial effects that:
the invention can increase the proliferation function of the cell in the expansion process of the mesenchymal stem cell, protect the differentiation function of the cell, and increase the differentiation function and speed of the cell in the differentiation process of the mesenchymal stem cell into the chondrocyte.
In the cell culture stage, the proliferation function of the mesenchymal stem cells cultured after adding vitamin D, vitamin A and EGF is obviously increased compared with the proliferation function of stem cells of a control group which is not added, and the proliferation function of the stem cells is obviously enhanced;
in the differentiation culture stage, mRNA expression of the induced mesenchymal stem cells after adding vitamin D, vitamin A and TGF-beta 1 is obviously increased compared with mRNA expression of type II collagen and proteoglycan of the mesenchymal stem cells among control groups which are not added;
the high-generation secondary mesenchymal stem cells induced by adding vitamin D, vitamin A and TGF-beta 1 can be differentiated into chondroblasts, the period of the induced differentiation of the mesenchymal stem cells into the chondroblasts is short, and the induced differentiation can be differentiated into the chondroblasts within 15 days.
The method for culturing the mesenchymal stem cells can also be applied to culturing the mesenchymal stem cells from other different sources;
the method can be used for establishing the mesenchymal stem cells of the main cell bank and the working cell bank and is applied to clinical tests.
Drawings
FIG. 1 is a P10 generation mesenchymal stem cell proliferation function determination; wherein ● denotes a control group, ■ denotes an experimental group 1, a-solidoid denotes an experimental group 2, a-X denotes an experimental group 3, diamond-solid denotes an experimental group 4, and O denotes an experimental group 5;
FIG. 2 shows the expression of glycosaminoglycan mRNA of mesenchymal stem cells after differentiation induction;
FIG. 3 shows the expression of type II collagen mRNA of mesenchymal stem cells after differentiation induction.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
The first embodiment is as follows: the method for promoting the mesenchymal stem cells to be differentiated into the chondroblasts comprises the following steps:
firstly, stem cell amplification culture:
recovering the umbilical cord source mesenchymal stem cells from P8 generation, culturing by adopting a serum-free culture medium, changing the culture solution the next day, continuously culturing the cells, wherein the culture medium used for changing the culture solution is a complete culture medium containing 0.8-1.5mol/L vitamin D, 1.5-2.5mol/L vitamin A and 90-110ng/ml EGF at the final concentration, digesting, passaging and finally obtaining the umbilical cord mesenchymal stem cells from P10 generation;
the function of the culture medium used for cell culture in the step is to promote the recovered cells to adhere to the wall and improve the activity of the cells.
Secondly, differentiation of chondroblasts:
taking the P10 substitute umbilical cord mesenchymal stem cells obtained by the culture in the step one, cleaning, sampling for cell counting, and adjusting the cell concentration to 1 × 104Per ml;
Changing the culture medium the next day, namely changing the culture medium for the first time, and carrying out differentiation culture, wherein the culture medium used for changing the culture medium for the first time is a differentiation basic culture medium containing 2mol/L vitamin D, 4mol/L vitamin A and 200ng/ml TGF-beta 1 at final concentration; the culture medium used for differentiation culture has the function of improving the differentiation function of stem cells.
Changing the culture medium every 3 days, wherein the culture medium used for changing the culture medium for the second time is a differentiation basal culture medium containing 2mol/L vitamin D, 4mol/L vitamin A and 400ng/ml TGF-beta 1, and the culture medium used for changing the culture medium for the second time is the same as the culture medium used for changing the culture medium for the second time; the second and later liquid change increases the concentration of TGF-beta 1, which is beneficial to promoting differentiation culture.
After 15 days of culture, differentiation of chondroblasts was completed.
The second embodiment is as follows: the first difference between the present embodiment and the specific embodiment is: and in the step one, digesting until the cell fusion degree reaches more than 70 percent to obtain single cells. The rest is the same as the first embodiment.
The reason why the digestion was carried out after the degree of cell fusion reached 70% or more was to increase the number of cells to a sufficient level.
Third embodiment the difference between the first embodiment and the second embodiment is that step one is performed according to 1 × 106Inoculating each cell/T175 flask, and carrying out passage after the cell fusion degree reaches more than 90%. The other is the same as in the first or second embodiment.
The passaging is carried out after the degree of cell fusion reaches 90% or more, in order to obtain a sufficient number of cells.
The fourth concrete implementation mode: the difference between this embodiment mode and one of the first to third embodiment modes is: and (3) carrying out immune marker detection on the umbilical cord mesenchymal stem cells obtained in the first step and the generation P10. The others are the same as in one of the first to third embodiments.
The immune marker detects surface antigens CD13, CD29, CD44, CD54, CD73, CD105, CD3, CD14, CD34, CD45 and endothelial cell surface antigen CD31, and aims to judge whether the cells have variation in the cell amplification process and meet the characteristics of mesenchymal stem cells.
The fifth concrete implementation mode: the difference between this embodiment and one of the first to fourth embodiments is: in the step one and the step two, the complete culture medium is the basic culture medium added with heparin sodium, and each 560mL of the basic culture medium is added with 120-. The other is the same as one of the first to fourth embodiments.
The effect of adding heparin sodium in the basic culture medium is to prevent the cells from aggregating and enable the cells to disperse and grow.
The sixth specific implementation mode: the difference between this embodiment and one of the first to fifth embodiments is: the differentiation basal medium in the second step is MesenCultMTHeparin sodium was added to the Osteogenic Differentiation Kit, each 560mL MesenCultMT120-160. mu.l heparin sodium was added to the Osteogenic Differentiation Kit. The other is the same as one of the first to fifth embodiments.
In MesenCultMTThe effect of adding heparin sodium in the Osteogenic Differentiation Kit is to prevent cell aggregation and enable cells to disperse and grow.
The seventh embodiment: the difference between this embodiment and one of the first to sixth embodiments is: step two, before inoculating cells in the 24-well plate, adding a slide, adding 450-. The other is the same as one of the first to sixth embodiments.
In the embodiment, a glass slide is arranged in the orifice plate, and the cells climb on the glass slide, so that the cells are convenient to store and observe under a mirror after being dyed. The saline removes gas between the slide and the well plate so that the slide is adjacent to the bottom of the well plate.
The following examples are given to illustrate the present invention, and the following examples are carried out on the premise of the technical solution of the present invention, and give detailed embodiments and specific procedures, but the scope of the present invention is not limited to the following examples.
Example 1:
firstly, stem cell amplification culture:
resuscitating the mesenchymal stem cells from the umbilical cord of the generation P8, culturing the cells in a sterile working table by adopting a serum-free culture medium, dividing the cells into an experimental group and a control group, wherein the experimental group is divided into an experimental group 1-an experimental group 5, changing the liquid the next day, continuously culturing the cells, digesting the cells by 0.125 percent of pancreatin after the cell fusion degree reaches more than 70 percent to obtain single cells, and culturing the cells according to the formula of 1 × 106Inoculating the single cell/T175 flask, and carrying out passage after the cell fusion degree reaches more than 90 percent to obtain the final productP10 passage cells. The culture medium used for cell culture in each group of the experimental group is different from that in the control group, and is specifically shown in table 2.
The immune marker detection of umbilical cord MSCs proves that the obtained P10 generation cells all express typical MSCs surface antigens CD13, CD29, CD44, CD54, CD73 and CD105, do not express hematopoietic stem cell surface antigens CD3, CD14, CD34, CD45 and endothelial cell surface antigen CD31, meet the characteristics of stem cells, and the P10 generation umbilical cord mesenchymal stem cells are seed cells for osteogenic differentiation.
Secondly, differentiation of chondroblasts:
taking the P10 generation umbilical cord mesenchymal stem cells obtained by the culture in the step one, washing the cells for 2 times by using normal saline, sampling the cells, counting the cells, and adjusting the cell concentration to 1 × 104Adding slide into 24-well plate, adding 500 μ l physiological saline, discarding physiological saline after 30min according to cell concentration of 1 × 104And (4) inoculating each hole, and culturing the cells. The culture medium used for cell culture in each group of the experimental group is different from that in the control group, and is specifically shown in table 2. The medium for cell culture in each group of this step was the same as that used for cell culture in step one.
The culture medium is changed the next day (namely, the first liquid change), and differentiation culture is carried out. The culture medium used for differentiation culture of the experimental group is different from that of the control group, and is specifically shown in table 2. The solution was changed every 3 days.
The experimental group and the control group are cultured for 21 days, the cells are rinsed once by PBS (phosphate buffer solution), toluidine blue staining is carried out for 30min, the cells are washed clean by triple-distilled water, hematoxylin is used for staining nuclei, absolute ethyl alcohol is used for dehydration, xylene is transparent, gelatin and glycerol are used for sealing, and the cells are observed under a mirror.
Example 2: quantitative detection of type II collagen and glycosaminoglycan:
the culture medium and cells of cartilage-inducing cells 7, 10, 13, 16, 19 and 21d of example 1 were collected for quantitative determination of type II collagen and glycosaminoglycan, respectively. The control group uses the P10 generation human umbilical cord mesenchymal stem cells with the same culture days. The collagen II quantitative detection adopts a hydroxyproline method, and each group of samples are operated according to the steps of hydroxyproline kit instructions (digestion method). The glycosaminoglycan is quantitatively determined by the Aliskiren method, each group of samples is digested by papain, the digestion product is added with the Aliskiron, and the absorbance value is measured at the 600nm wavelength of an enzyme-labeling instrument.
Example 3: and (3) real-time quantitative PCR detection:
and carrying out real-time quantitative PCR detection on the P10 generation human umbilical cord mesenchymal stem cells and the induced and differentiated chondrocytes. 300ng of total RNA was reverse transcribed into cDNA as template for q PCR. Real-time quantitative PCR is used for detecting mRNA expression of type II collagen and glycosaminoglycan, and GADPH is used as an internal reference.
Identifying the induced mesenchymal stem cells by qRT-PCR: and extracting total RNA of the induced mesenchymal stem cells by TRIzol, carrying out reverse transcription to obtain cDNA, and carrying out qRT-PCR reaction. The primer sequences are shown in table 1:
TABLE 1
Normal mesenchymal stem cells were used as a control group in the PCR reaction. According to the amplification efficiency of type II collagen and glycosaminoglycan genes and Ct value given by a fluorescence quantitative PCR instrument in qRT-PCR, GADPH is taken as an internal reference gene, and RT-PCR results are applied 2-ΔΔCtThe method is used for analysis, and the expression quantity of each gene is calculated according to a formula:
Δ Δ Ct ═ treatment group (Ct)Target gene-CtGAPDH) Control group (Ct)Target gene-CtGAPDH)
The difference multiple of the expression level of the target gene is 2-ΔΔCt
TABLE 2
| Grouping | Cell culture | Differential culture |
| Control group | Complete culture medium | Differentiation basal medium |
| |
Complete Medium + VD+VA+EGF | Differentiation basal Medium + VD+VA+TGF-β1 |
| |
Complete Medium + VD+VA | Differentiation basal Medium + VD+VA |
| Experimental group 3 | Complete Medium + VD+EGF | Differentiation basal Medium + VD+TGF-β1 |
| |
Complete Medium + VA+EGF | Differentiation basal Medium + VA+TGF-β1 |
| |
Complete Medium + EGF | Differentiation basal Medium + TGF- |
Cell culture: the culture medium used for cell culture of the control group is a complete culture medium; the complete culture medium is prepared by adding heparin sodium into a basic culture medium, and 160 mu l of heparin sodium is added into every 560mL of the basic culture medium. Experimental groups 1 to 5 groups were each divided into groups according to Table 2, and the groups were added to the medium at a final concentration of 1mol/L for vitamin D, 2mol/L for vitamin A, and 100ng/ml for EGF.
Differentiation culture: the control group was cultured using a differentiation basal medium; the differentiation basal medium is MesenCultMTHeparin sodium was added to the Osteogenic Differentiation Kit, each 560mL MesenCultMT160 mu L of heparin sodium is added into the Osteogenic Differentiation Kit, the experimental groups 1-5 are respectively divided into groups according to the table 2, the added components are respectively added into culture media, the cartilage culture medium in the experimental group of the first liquid change contains 2mol/L of vitamin D, 4mol/L of vitamin A and 200ng/ml of TGF- β 1, the second liquid change and the subsequent liquid change, the culture media in the experimental groups 1-5 contain 2mol/L of vitamin D, 4mol/L of vitamin A and 400ng/ml of TGF- β 1.
The experimental results are as follows:
the immunophenotypic expression rate of P10 generation cells obtained by stem cell expansion culture is shown in Table 3.
The detection result of the flow cytometry shows that the cells of each experimental group are not mutated in the cell amplification process after the components are added into the culture medium, and the characteristics of the mesenchymal stem cells are met.
Table 3 immunophenotypic expression rate (%):
| cells | CD34 | CD45 | CD73 | CD90 | CD105 |
| Control group | 0.58±0.06 | 0.29±0.08 | 99.43±0.02 | 99.92±0.03 | 99.71±0.02 |
| |
0.29±0.09 | 0.31±0.01 | 99.71±0.05 | 99.98±0.06 | 99.86±0.04 |
| |
0.19±0.06 | 0.34±0.08 | 99.61±0.09 | 99.65±0.03 | 99.34±0.09 |
| |
0.22±0.02 | 0.29±0.06 | 99.39±0.06 | 93.97±0.09 | 99.65±0.05 |
| |
0.30±0.01 | 0.21±0.03 | 99.54±0.08 | 95.37±0.05 | 99.62±0.04 |
| |
0.24±0.03 | 0.34±0.04 | 99.32±0.06 | 96.34±0.04 | 99.34±0.06 |
The doubling time measurements of cells cultured in different media are shown in table 4. The result of detecting the proliferation function of the mesenchymal stem cells of the P10 generation is shown in figure 1. From experimental data, it is found that the cell growth after culture in different media has different doubling times, and that the shorter the time, the faster the cell doubling and the higher the growth function. The experimental group 1 has the shortest doubling time, which indicates that the vitamin D, the vitamin A and the EGF are simultaneously added into the culture medium for culturing the cells of the experimental group 1, and the enhancement of the cell proliferation function is facilitated. The three components of vitamin D, vitamin A and EGF are interacted, have synergistic effect and are all indispensable.
TABLE 4 cell proliferation function assay
| Cells | Doubling Time (DT) |
| Control group | 27.8±2.2 |
| |
21.2±2.3 |
| |
23.8±2.1 |
| |
26.8±1.9 |
| |
24.5±1.8 |
| |
24.6±2.2 |
After 21 days of chondroblast differentiation culture, the expression changes of glycosaminoglycan mRNA and type II collagen mRNA of the mesenchymal stem cells are shown in Table 5. Glycosaminoglycan and type II collagen are continuously produced proteins in the chondrocyte differentiation process, and experimental results show that in 5 experimental groups, after different factors are respectively added into a culture medium, the cells in the experimental group 1 have the best differentiation function and the highest expression level. The experimental group 1 is proved that the culture medium for differentiation culture is simultaneously added with vitamin D, vitamin A and EGF, which is beneficial to the enhancement of the cell proliferation function. The three components of vitamin D, vitamin A and TGF-beta 1 interact with each other, have synergistic effect and are all indispensable.
TABLE 5 changes in expression of glycosaminoglycan mRNA and type II collagen mRNA of mesenchymal stem cells after induction
Claims (7)
1. A method for promoting differentiation of mesenchymal stem cells into chondroblasts, the method comprising the steps of:
recovering umbilical cord-derived mesenchymal stem cells from P8 generation, culturing by adopting a serum-free culture medium, changing the culture solution the next day, digesting and passaging to finally obtain umbilical cord mesenchymal stem cells from P10 generation, wherein the culture medium used for changing the culture solution is a complete culture medium containing 0.8-1.5mol/L vitamin D, 1.5-2.5mol/L vitamin A and 90-110ng/ml EGF at final concentration;
secondly, taking the P10 generation umbilical cord mesenchymal stem cells obtained by the culture in the step one, cleaning, sampling for cell counting, and adjusting the cell concentration to 1 × 104Per ml;
cell concentration 1 × 10 in 24-well plates4Inoculating P10 generation umbilical cord mesenchymal stem cells per hole, and performing cell culture by using a complete culture medium containing 0.8-1.5mol/L vitamin D, 1.5-2.5mol/L vitamin A and 90-110ng/ml EGF at final concentration;
changing the culture medium the next day, namely changing the culture medium for the first time, and performing differential culture; the culture medium used for the first liquid change is a differentiation basal culture medium containing 1.8-2.2mol/L vitamin D, 3.5-4.5mol/L vitamin A and 190-210ng/ml TGF-beta 1;
then changing the culture medium every 3 days, wherein the culture medium used for changing the culture medium for the second time is a differentiation basic culture medium containing 1.8-2.2mol/L vitamin D, 3.5-4.5mol/L vitamin A and 390-410ng/ml TGF-beta 1, and the culture medium used for changing the culture medium is the same as the culture medium used for changing the culture medium for the second time;
and culturing for 13-15 days to complete the differentiation culture of the chondroblasts.
2. The method for promoting differentiation of mesenchymal stem cells into chondroblasts according to claim 1, wherein in the first step, the cells are digested until the degree of cell fusion reaches more than 70%, and single cells are obtained.
3. The method for promoting differentiation of mesenchymal stem cells into chondroblasts according to claim 1 or 2, wherein the step one is performed according to 1 × 106Inoculating each cell/T175 flask, and carrying out passage after the cell fusion degree reaches more than 90%.
4. The method for promoting differentiation of mesenchymal stem cells into chondroblasts according to claim 3, wherein the umbilical cord mesenchymal stem cells of P10 generation obtained in the first step are subjected to immunolabeling detection.
5. The method for promoting differentiation of mesenchymal stem cells into chondroblasts according to claim 1 or 2, wherein the complete medium in the first step and the second step is the basic medium supplemented with heparin sodium, and each 560mL of the basic medium is supplemented with 160. mu.L of heparin sodium.
6. The method for promoting differentiation of mesenchymal stem cells into chondroblasts according to claim 1 or 2, wherein the differentiation basic medium in step two is MesenCultMTHeparin sodium was added to the Osteogenic Differentiation Kit, each 560mL MesenCultMT120-160. mu.l heparin sodium was added to the Osteogenic Differentiation Kit.
7. The method for promoting differentiation of mesenchymal stem cells into chondroblasts according to claim 5, wherein in the second step, before seeding cells in a 24-well plate, a glass slide is added, 500 μ l of physiological saline is added, and the physiological saline is discarded after 30-35 min.
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