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目的:探讨体外心肌微环境对骨髓间充质干细胞(BMSCs)分化为心肌样细胞的诱导作用。方法:分离大鼠骨髓间充质干细胞(BMSCs)和心肌细胞并在体外培养纯化。以2×10~4/mL的细胞密度种植心肌细胞,培养72h后收集上清,在BMSCs的培养基内加入心肌细胞培养上清诱导BMSCs分化为心肌样细胞,实验共分6组:10%、20%、30%、40%及50%心肌细胞培养上清组及含10%胎牛血清的对照组,均诱导BMSCs7d,每天在倒置显微镜下观察细胞的形态变化;应用免疫细胞化学技术检测不同浓度心肌细胞培养上清组诱导7d后BMSCs中a-平滑肌肌动蛋白(a-sMA)、β-肌动蛋白(β-actin)、心肌特异性肌钙蛋白T(Troponin-T)、CD31以及FactorⅧ的表达。结果:心肌细胞培养上清组诱导BMSCs7d后,细胞的形态没有改变。10%、20%、30%、40%及50%心肌细胞培养上清组a-sMA、。-actin以及Troponin-T的表达均呈阳性,以30%心肌细胞培养上清组的蛋白表达量最高(P<0.01),而CD31和FactorⅧ的表达呈阴性。结论:心肌细胞的培养上清能够诱导BMSCs分化为心肌样细胞,且30%心肌细胞培养上清是诱导BMSCs向心肌样细胞分化的最适浓度。
AIM: To investigate the induction of myogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into cardiomyocytes by extracorporeal myocardial microenvironment. Methods: Rat bone marrow mesenchymal stem cells (BMSCs) and cardiomyocytes were isolated and cultured in vitro. The cardiomyocytes were seeded at a density of 2 × 10 ~ 4 / mL. The supernatants were collected 72h after culture. Cardiomyocytes were cultured in BMSCs medium to induce cardiomyocyte-like cells to differentiate into BMSCs. The experiment was divided into 6 groups: 10% BMSCs were induced in 7 days, 20%, 30%, 40% and 50% of cardiomyocyte culture supernatant and control group containing 10% fetal bovine serum respectively. Morphological changes of cells were observed under inverted microscope every day. Immunocytochemistry The a-sMA, β-actin, Troponin-T, B lymphocyte subsets in BMSCs after induced by different concentrations of cardiomyocyte culture supernatants for 7 days, And Factor VIII expression. Results: The morphological changes of BMSCs were not observed after 7 days induction of cardiomyocyte culture supernatant. A-sMA, 10%, 20%, 30%, 40% and 50% of cardiomyocyte culture supernatant group. The expression of -actin and Troponin-T were all positive. The expression of CD31 and Factor VIII was the highest in 30% of cardiomyocyte culture supernatants (P <0.01). CONCLUSION: The culture supernatant of cardiomyocytes can induce the differentiation of BMSCs into cardiomyocyte-like cells, and 30% of cardiomyocyte culture supernatant is the optimal concentration for inducing BMSCs to differentiate into cardiomyocyte-like cells.