摘要
目的构建人survivin特异性siRNA,探讨其对骨肉瘤细胞MG63细胞周期及survivin表达的影响。方法构建sur-vivin特异性的RNA干涉载体,转染MG63细胞,G418筛选稳定转染的细胞系,以Westernblot法检测survivin蛋白表达变化,透射电镜、Hoechst染色检测细胞凋亡情况,流式细胞仪检测细胞周期变化。结果成功构建了survivin基因siRNA真核表达载体PsiS,获得了稳定转染的细胞系。与正常MG63细胞、阴性对照细胞相比,MG63/PsiS细胞中survivin蛋白表达减少,部分细胞核致密浓染或碎块状浓染,凋亡率增加了7倍,而G2/M期细胞减少近50%。结论特异性siRNA能够明显抑制survivin基因在MG63细胞中的表达,抑制细胞增殖,促进细胞凋亡。
Objective To explore the effects of siRNA on survivin gene expression in osteosarcoma cell line MG63 using siRNA eukaryotie expression vector, and on the cell cycle of osteosarcoma. Method The specific RNA interference （RNAi） fragments targeting survivin gene were synthesized, and they were cloned into pSilencer 3. 0-H1 neo plasmid vector. After the vector was constructed, MG63 cells were transfected with negative control vector or RNAi vectors and selected by G418. Expression of protein of survivin in the stable transfected cells was determined by Western blot. These cells which were cultured on cover slips were observed with electron microscopy. Apoptosis analysis was performed with flow cytometry and Hoechst staining. Results Specific siRNA eukaryotic expression vector PsiS targeting survivin gene was constructed successfully. The stable transfectants containing negative control vector and PsiS were obtained. Protein expression of survivin was inhibited significantly in MG63/PsiS cells, whereas survivin gene expression levels were hardly changed in normal MG63 and negative control cells. There were much more pyknotic nuclei found in MG63/PsiS pool than those in MG63 and control. Analysis of DNA content in PsiS transfectants revealed a 7-fold increase in the fraction of sub-G1 peak （apoptotic peak） as compared with other transfectants under the same experimental conditions （P〈0. 01）. This was also associated with a 50% decrease in the G2/M fraction. Conclusion siRNA of survivin could effectively knock down survivin expression in MG63 cells, inhibit tumor cells proliferation and increase cell apoptosis significantly.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2007年第2期104-106,共3页
Medical Journal of Chinese People's Liberation Army
