丹参诱导骨髓基质细胞神经元样细胞分化的microRNA-128路径

目的　研究microRNA-128在丹参制剂诱导大鼠骨髓基质细胞(bone marrow strowal cells，BMSCs)向神经元样细胞分化过程中的调控作用。  方法　培养鉴定大鼠BMSCs，应用反义寡居核苷酸转染技术（Lipofectamin 2000）转染microRNA-128 inhibitors于BMSCs；取转染和未转染的BMSCs，待细胞增殖到60%~70%融合时进行预诱导分化，预诱导24 h后，进行诱导分化；根据处理方式不同分为4组：未诱导组（A组），丹参制剂诱导组（B组），转染未诱导组（C组），转染诱导组（D组）。流式细胞术鉴定BMSCs；qPCR检测microRNA-128及神经元特异性烯醇化酶（NSE）、巢蛋白（Nestin）、微管蛋白（β3-tubulin）的mRNA表达；Western blot检测NSE、Nestin和β3-tubulin的蛋白表达。  结果    BMSCs流式细胞检测CD29和CD90双阳性率达99.17%，CD11b和CD45双阴性率达99.21%；丹参制剂诱导及microRNA-128 inhibitors转染后，细胞microRNA-128表达量显著降低（P<0.05）；诱导与未诱导组，转染与未转染组比较，NSE、Nestin和β3-tubulin的mRNA和蛋白表达均显著升高（P<0.05）。  结论　丹参制剂能够抑制BMSCs的microRNA-128表达，进而影响BMSCs神经分化相关蛋白的表达，促进BMSCs向神经元样细胞分化。

MicroRNA-128 regulates differentiation of bone marrow strowal cells induced by Salvia miltiorrhiza

Objective　To study the regulation of microRNA-128 on differentiation of rat BMSCs into neuron-like cells induced by Salvia miltiorrhiza.  Methods　Rat BMSCs were isolated, purified and identification in vitro. When the cells reached 80% confluence, microRNA-128 inhibitors were transfected by Lipofectamin 2000. Transfected and untransfected cells were pre-induced for 24h. Then the pre-induced cells induced for 5h by Salvia miltiorrhiza. According to the above experiment, cells were divided into four groups: a control group, an induced group, a transfected group, and an induced-transfected group. To identify the phenotypes, flow cytometry analysis was performed. The expression of microRNA-128 and mRNA for NSE, Nestin and β3-tubulin was detected by qPCR. NSE, Nestin and β3-tubulin protein expression were determined by western blot.　Results　Most cells showed the characteristic phenotypes of BMSCs positive for CD29 and CD90 (99.17%) and negative for CD11b and CD45 (99.21%). The microRNA-128 expression of the induced group and the transfected group were found to be significantly higher than the control group. The mRNA and protein expression of NSE, Nestin and β3-tubulin were found to be significantly higher in the induced group and in the transfected group (P<0.05).　Conclusions　Salvia miltiorrhiza can inhibit microRNA-128 expression of BMSCs to increase neural marker protein expression to promote differentiation of BMSCs into neuron-like cells.