Notch-1在骨髓间质干细胞分化为神经细胞中的作用

目的:采用RNA干扰(RNA interference,RNAi)技术,抑制Notch-1基因的表达,从而观察Notch-1基因在骨髓间质干细胞(MSCs)分化为神经细胞后的存活及分化中的作用.方法:构建mNotch-l短发夹状RNA(short hairpin RNA,mNotch-1shRNA);然后对mNotch-lshRNA转染的MSCs进行形态学观察;MTT检测转染前、转染24h、转染3天的MSCs的存活率;体外条件下采用BDNF、全反式维甲酸等诱导mNotch-lshRNA转染的MSCs分化,采用免疫细胞化学染色,检测Nestin、NSE及NF200(神经元标记物)、GFAP(神经胶质细胞标记物)的表达.结果:mNotch-lshRNA转染成功的细胞立体感增强,Notch-1基因表达消失,转染后24h和3天的MSCs细胞存活率下降,与未转染和转染对照组比较有显著性差异(P＜0.01);转染mNotch-1shRNA的MSCs向神经细胞的分化效率显著提高,NSE、NF200的表达均高于未转染的和转染对照组的,且未见GFAP表达.结论:mNotch-lshRNA抑制Notch-1基因的表达,促进MSCs向神经细胞的分化效率,提示MSCs的横向分化过程可能与神经干细胞类似,阻断Notch信号通路,会加MSCs向神经细胞分化的效果.

Role of Notch- 1 in the Differentiation of Rat Mesenchymal Stem Cells into Neurons

Objectives; To identify the roles of Notch - 1 gene for survivaland differentiation of mesenchymal stem cells ( MSCs) into neurons by way of RNA interference ( RNAi) technique to suppress expression of Notch - 1 gene. Methods; Firstly, construct mNotch - 1 short hairpin RNA (mNotch -IshRNA) and then to oberve configuration of MSCs transfected by mNotch - IshRNA; MTT was employed to evaluate the survival ratio of MSCs before transfection, 24 hours and 3 days after transfection, respectively; agents, such as BDNF, retinoid acid, were obtained to induce MSCs transfected by mNotch - IshRNA in vitro and expression of Nestin, NSE and NF200( neuron marker agent) and GFAP( neural glial cell marker agent) were determined by immunocytochemistry stain technique. Results: The cell cubic configuration was enforced and expression of notch - 1 gene vanished by transfection of mNotch -IshRNA. The survival ratio of MSCs by transfection decreased and there was significant differences between non - ransfection and transfection - controlling group after 24 hours and 3 days of transfection ( P <0. 01). The differentiation efficacy of MSCs transfected by mNotch - IshRNA into neurons and the expression of NSE,NF200 were increased significantly than non - ransfection and transfection -controlling group, and there was no expression of GFAP. Conclusions; The differentiation efficacy of MSCs can be enforced by mNotch - IshRNA to suppress Notch - 1 expression, which implies that the transverse differentiation of MSCs is similar to that of neural stem cells. The blocking of Notch - 1 signal pathway might promote differentiation of MSCs into neural stem cells.