CN111534486A - Application of active ingredient to inducing chondrogenic differentiation of mesenchymal stem cells and chondrogenic differentiation induction culture medium - Google Patents
Application of active ingredient to inducing chondrogenic differentiation of mesenchymal stem cells and chondrogenic differentiation induction culture medium Download PDFInfo
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- CN111534486A CN111534486A CN202010570222.2A CN202010570222A CN111534486A CN 111534486 A CN111534486 A CN 111534486A CN 202010570222 A CN202010570222 A CN 202010570222A CN 111534486 A CN111534486 A CN 111534486A
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Abstract
The invention discloses an application of an active ingredient in inducing bone marrow mesenchymal stem cells to chondrogenic differentiation and a chondrogenic differentiation induction culture medium. The invention discovers that the inositol nicotinate has the function of inducing the differentiation of the human mesenchymal stem cells hBMSCs to cartilage cells in vitro and can also promote the in vitro proliferation of the human mesenchymal stem cells hBMSCs, so that the inositol nicotinate can be used for preparing a culture medium for promoting the proliferation of the mesenchymal stem cells and inducing the chondrogenic differentiation of the mesenchymal stem cells.
Description
Technical Field
The invention belongs to the field of biological stem cells, relates to stem cell induced differentiation, and particularly relates to an application of an active ingredient in inducing bone marrow mesenchymal stem cells to chondrogenic differentiation and a chondrogenic induced differentiation culture medium.
Background
Human mesenchymal stem cells (hBMSCs) are adult stem cells having multipotent differentiation potential and can be induced to differentiate into osteoblasts, adipocytes, chondrocytes, and neurons. hBMSCs have the advantages of easy availability of materials, easy in vitro expansion, and long-term maintenance of multi-differentiation potential, and can be autotransplanted without problems of tissue matching and immune rejection, because they are considered to be the best seed cells in cartilage tissue engineering.
The literature, "study on the effect and mechanism of Adenosine triphosphate on the differentiation of mesenchymal stem cells into chondrocytes" discusses the effect and mechanism of Adenosine Triphosphate (ATP) on the differentiation of mesenchymal stem cells (BMSCs) into chondrocytes, and finds that a certain concentration of ATP has a promoting effect on the differentiation of BMSCs into chondrocytes, and may play a role by mediating P2X7 receptor (journal of tissue engineering and reconstructive surgery, 2019).
Literature "in vitro induction of bone marrow mesenchymal stem cells directed differentiation into chondrocytes: the synergistic stimulation effect of transforming growth factor beta 1 and insulin-like growth factor 1 analyzes the synergistic stimulation effect of transforming growth factor beta 1 and insulin-like growth factor 1 in vitro induced bone marrow mesenchymal stem cells to directionally differentiate into cartilage cells so as to obtain the optimal induction effect, and finds that when the bone marrow mesenchymal stem cells are induced in vitro to differentiate into the cartilage cells, the transforming growth factor beta 1 and the insulin-like growth factor 1 have the synergistic stimulation effect, and the better induction effect can be obtained by combining the transforming growth factor beta 1 and the insulin-like growth factor 1 (Chinese tissue engineering research, 2018).
The research on the effect of icariin on the induction and differentiation of human mesenchymal stem cells into chondrocytes shows that icariin can induce hBMSCs to differentiate into chondrocytes in vitro (New Chinese medicine, 2016).
Since hBMSCs have many advantages as seed cells, research on induced differentiation into chondrocytes is still on the way.
Disclosure of Invention
The invention aims to provide an application of an active ingredient in inducing bone marrow mesenchymal stem cells to chondrogenic differentiation and a chondrogenic differentiation induction culture medium, wherein the active ingredient is inositol nicotinate.
The purpose of the invention is realized by the following technical scheme:
an application of an active component in vitro inducing the chondrogenic differentiation of bone marrow mesenchymal stem cells, wherein the component is inositol nicotinate.
Application of inositol nicotinate in preparing culture medium for inducing bone marrow mesenchymal stem cells to form cartilage.
A culture medium for promoting the proliferation of bone marrow mesenchymal stem cells and inducing the chondrogenic differentiation of the bone marrow mesenchymal stem cells comprises a basic culture medium and an active ingredient inositol nicotinate.
Has the advantages that:
the invention discovers that the inositol nicotinate has the function of inducing the differentiation of the human mesenchymal stem cells hBMSCs to cartilage cells in vitro and can also promote the in vitro proliferation of the human mesenchymal stem cells hBMSCs, so that the inositol nicotinate can be used for preparing a culture medium for promoting the proliferation of the mesenchymal stem cells and inducing the chondrogenic differentiation of the mesenchymal stem cells.
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FIG. 1 shows toluidine blue staining results;
FIG. 2 shows the result of Western Blot detection.
Detailed Description
The following examples are presented to illustrate the essence of the present invention, but should not be construed as limiting the scope of the present invention.
First, experimental material
Human mesenchymal stem cells hBMSCs were purchased from ScienCell, USA.
Fetal bovine serum and L-DMEM medium were from Gibco, USA, and pancreatin was purchased from Biyunyan.
BCA kit was purchased from petunia. The purity of the inositol nicotinate is not less than 98%.
The low temperature refrigerator is a Thermo Fisher Scientific product.
The cell culture box is a Thermo Fisher Scientific product.
Second, Experimental methods
1. In vitro culture of hBMSCs
After the human mesenchymal stem cells hBMSCs are recovered, the human mesenchymal stem cells are cultured in L-DMEM medium (namely complete medium) containing 10 percent fetal calf serum, 100U/ml penicillin and 100 mu g/ml streptomycin at 37 ℃ and 5 percent CO2Culturing under conditions, replacing the culture medium for 2-3 days, growing the cells to 70-80% confluence state, digesting with pancreatin, and carrying out passage.
2. Grouped and chondrogenic induction
Taking human mesenchymal stem cells hBMSCs with the fusion rate of about 80-90% after recovery and passage, centrifuging, discarding the culture solution, adding 0.25% pancreatin for digestion at 37 ℃, adding an L-DMEM medium with the same volume for stopping digestion, centrifuging, discarding the culture solution, and resuspending with a complete culture medium to prepare the hBMSCs with the cell concentration of 2 × 105/mL cell suspension, seeded in 6 well plates at 2 mL/well, 5% CO at 37 ℃2Culturing in constant temperature incubator for 12 hr, changing the inducing group to complete culture medium containing 5 μ M inositol nicotinate, changing the control group to new complete culture medium, and continuing to culture at 37 deg.C and 5% CO2Culturing for 14d in a constant-temperature incubator, replacing the corresponding culture medium every 2-3 d, and repeating 3 holes in each group.
3. Toluidine blue staining detection of chondrocyte marker glycosaminoglycan
Washing the cell slide cultured for 14d with PBS (phosphate buffer solution), fixing paraformaldehyde with mass concentration of 40g/L at normal temperature for 1h, and washing with distilled water for 15 min; toluidine blue was added to cover the cells and stained for 4h at room temperature. Removing stock solution, decolorizing with anhydrous ethanol, washing with distilled water until water is colorless, and sealing with neutral gum. Observing under an inverted microscope, and collecting slices.
4. Western Blot for detecting the expression of chondrogenic differentiation related proteins SOX9 and COL II
Collecting the human bone marrow mesenchymal stem cells hBMSCs of the induction group and the control group which are cultured for 14d, washing with PBS, cracking on ice, extracting total protein, and determining the protein concentration by using a BCA kit. Performing SDS-PAGE electrophoresis and membrane conversion on equivalent protein, sealing for 1h, adding primary antibodies of anti-SOX 9, COL II and GAPDH, shaking overnight at 4 ℃, adding a secondary antibody marked by HRP, incubating for 1h at room temperature, performing chemiluminescence for color development, and collecting images by a gel imager.
5. MTT method for determining influence of inositol nicotinate on in-vitro proliferation activity of hBMSCs
Taking human mesenchymal stem cells hBMSCs with the fusion rate of about 80-90% after recovery passage, centrifuging, discarding the culture solution, adding 0.25% pancreatin for digestion at 37 ℃, adding an L-DMEM medium with the same volume for stopping digestion, centrifuging, discarding the culture solution, and resuspending with the whole culture medium to obtain the cells with the concentration of 1 × 105The cell suspension was seeded in 96-well plates at 100. mu.L/well in 5% CO at 37 ℃%2Culturing in constant temperature incubator for 12 hr, changing the inducing group to complete culture medium containing 5 μ M inositol nicotinate, changing the control group to new complete culture medium, and continuing to culture at 37 deg.C and 5% CO2After 48h incubation in a constant temperature incubator, 20. mu.L of 5g/L MTT solution was added to each well, incubation was continued for 4h, and the supernatant was discarded for each wellAdding 150 mu L of dimethyl sulfoxide into the wells, mixing uniformly, detecting the absorbance value at 490nm of an enzyme labeling instrument, taking the OD value of a control group as the proliferation rate of 100%, and calculating according to the 'OD value of an induction group/OD value of the control group of × 100%' to obtain the proliferation rate of the induction group, wherein each group comprises 5 multiple wells.
6. Statistical treatment
The experimental data are all expressed as mean ± standard deviation, and the difference between groups is compared by t-test.
Third, experimental results
1. Toluidine blue staining results
The results are shown in FIG. 1, the toluidine blue staining of the control group is negative, and the cytoplasmic staining of the slide cell is not obvious; the induced tolidine blue is positive in staining, the nucleus of the reptile cell is dark blue, the cytoplasm is bluish purple, and the staining is obvious. Toluidine blue staining is a method for detecting a marker glycosaminoglycan in chondrocytes, the staining result is positive if the glycosaminoglycan exists, and the total content of the glycosaminoglycan is an important biochemical index representing the chondrogenic differentiation level of hBMSCs.
2. Western Blot detection result
The results are shown in FIG. 2, which induces positive expression of the constitutive cartilage differentiation associated proteins SOX9 and COL II, but no significant expression was observed in the control group. SOX9 is a high-mobility group protein transcription factor, is a marker of the early chondrogenic differentiation stage of mesenchymal progenitor cells, SOX9 not only can bind and activate a chondrogenic enhancer sequence of non-cartilage tissue cells to enable the cartilage tissue cells to be in a cartilage cell phenotype, but also can interact with Wnt/beta-catenin and TGF-beta/Smad signal transduction pathways to regulate the differentiation of cartilage cells. COL II is the main structural component of chondrocytes, maintains the basic skeleton of cells, is also a marker protein of chondrocytes, and is mostly used for measuring the number and the mature state of the chondrocytes in clinical research, so that the COL II and the marker protein are important markers of the chondroblasts.
3. MTT method measurement results
The results are shown in table 1, the cell proliferation rate of the induced group is significantly higher than that of the control group, which indicates that the inositol nicotinate can also significantly promote the in vitro proliferation of the hBMSCs.
TABLE 1 proliferation Rate of the cells of each group
The experiment shows that the inositol nicotinate has the function of inducing the differentiation of the human mesenchymal stem cells hBMSCs to cartilage cells in vitro and can also promote the proliferation of the human mesenchymal stem cells hBMSCs in vitro, so that the inositol nicotinate can be used for preparing the culture medium for promoting the proliferation of the mesenchymal stem cells and inducing the chondrogenic differentiation of the mesenchymal stem cells.
The above examples are provided to illustrate the essence of the present invention, but should not be construed as limiting the scope of the present invention.
Claims (3)
1. An application of an active component in vitro inducing the chondrogenic differentiation of bone marrow mesenchymal stem cells, wherein the component is inositol nicotinate.
2. Application of inositol nicotinate in preparing culture medium for inducing bone marrow mesenchymal stem cells to form cartilage.
3. A culture medium for promoting the proliferation of bone marrow mesenchymal stem cells and inducing the chondrogenic differentiation of the bone marrow mesenchymal stem cells comprises a basic culture medium and an active ingredient inositol nicotinate.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111961643A (en) * | 2020-08-26 | 2020-11-20 | 杭州优渡生物科技有限公司 | Induction method for differentiation of stem cells into chondrocytes |
| CN113841690A (en) * | 2021-11-17 | 2021-12-28 | 李广超 | Protective agent for CAR-T cell cryopreservation and cryopreservation method |
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2020
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111961643A (en) * | 2020-08-26 | 2020-11-20 | 杭州优渡生物科技有限公司 | Induction method for differentiation of stem cells into chondrocytes |
| CN113841690A (en) * | 2021-11-17 | 2021-12-28 | 李广超 | Protective agent for CAR-T cell cryopreservation and cryopreservation method |
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Application publication date: 20200814 |