Application of stx1b gene in promoting growth and differentiation of human adipose-derived mesenchymal stem cells
A technology of mesenchymal stem cells and fat, applied in the field of STX1B gene in promoting the growth and differentiation of human adipose mesenchymal stem cells
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Embodiment 1
[0024] Example 1 Isolation and culture of adipose-derived mesenchymal stem cells
[0025]Take about 300 mg of adipose tissue, wash it repeatedly with PBS containing 200 U / mL penicillin and 200 mg / L streptomycin, cut it into a paste with ophthalmic scissors, transfer it into a culture bottle, add 3 times the volume of 0.1% type Ⅰ collagenase, and keep at 37 °C Shake and digest in a constant temperature water bath for 70 minutes. At this time, the liquid surface is divided into three layers, the upper layer is a yellow oily fat cell layer, the middle layer is adipose tissue layer, and the lower layer is a collagenase layer containing mononuclear cells. Centrifuge at 1500r / min for 15min, discard the supernatant, add complete culture medium (HG-DMEM medium containing 10% fetal bovine serum and 100× double antibody by volume fraction), pipette the cell pellet, filter through a 200-mesh cell sieve, and store at 37°C. The volume fraction was 5% CO2 saturated humidity condition. Afte...
Embodiment 2
[0026] Example 2 Induction experiment of adipose-derived mesenchymal stem cells into nerve cells
[0027] When the 3rd generation adipose stem cells were confluent to 80%, the complete medium was discarded, the control group continued to add complete medium, and the induction method used in the experimental group was: adding human nerve induction solution: 10ug / L EGF, 20ug / L bFGF and 10ug / L BDNF, after 3 days, nerve inducers were added: 120umol / L indomethacin, 3mg / L insulin, 300umol / LIBMX. After 48 hours of adding the nerve inducer, it was observed that ADSCs differentiated into nerve cells, the liquid in the 6-well plate was discarded, and washed twice with PBS, 5 minutes each time. Add the same permeabilizing agent (methanol: acetone = 1:1), wash with PBS 3 times after 30 min, 5 min each time. Then add anti-GFAP primary antibody (diluted at 1:50), anti-β-tubulin III primary antibody (diluted at 1:50), add the same amount of PBS to the control group, incubate overnight at 4...
Embodiment 3
[0031] Example 3 Bioinformatics analysis and screening of nerve cell-related genes
[0032] The applicant performed expression profile analysis on the cells obtained before the differentiation of human adipose-derived mesenchymal stem cells into neural cells and after induction in Example 2. First, the expression value of the original gene was obtained, and the data was analyzed by R software. The final data was obtained with the limma function package The differentially expressed genes before and after differentiation were analyzed, and the determined differential analysis conditions were that the gene expression multiple was greater than 1 times, and the FDR Xiaoyu was 0.01. Through analysis and final screening, the two most significantly differentially expressed genes induced by differentiation regulators were obtained, as shown in Table 1.
[0033] Table 1 Differentially expressed genes
[0034] Gene fold change Homosapiens nudixhydrolase 12 (NUDT12) 3.2...
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