丹参诱导骨髓基质细胞神经元样细胞分化的microRNA-128路径
黄涛, 张志强, 余磊, 谢才军, 祝栋安, 彭子壮, 陈捷晗
中国临床解剖学杂志 ›› 2014, Vol. 32 ›› Issue (4) : 427-431.
丹参诱导骨髓基质细胞神经元样细胞分化的microRNA-128路径
MicroRNA-128 regulates differentiation of bone marrow strowal cells induced by Salvia miltiorrhiza
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。
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.
丹参制剂 / 骨髓基质细胞 / microRNA-128 / 神经元 / 细胞分化
Salvia miltiorrhiza / BMSCs / microRNA-128 / Neuron / Cell differentiation
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广东省自然科学基金(S2011010005403);广东省科技计划项目(粤科函社字[2010]1096号)
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