【摘要】 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。更多还原

【Abstract】 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 24 h. 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 CD11 b 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.更多还原