摘要
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-1短发夹状RNA(short hairpin RNA,mNotch -1 shRNA);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-lshRNA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。
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 -1shRNA; 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 -1shRNA 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 -1shRNA. 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 - 1 shRNA into neuroils 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.
出处
《重庆医科大学学报》
CAS
CSCD
2005年第6期781-786,共6页
Journal of Chongqing Medical University
基金
重庆市卫生局科学基金(03-2-070)重庆市卫生局中医处科学基金(2004-B-064)中国博士后科学基金项目(第29期
2003033363)。
