人存活素特异性siRNA促进SOSP-9607细胞凋亡的实验研究  

Human survivin targeted siRNA enhances osteosarcoma cell SOSP-9607 apoptosis

作　　者：孙宏慧[1] 范清宇[1] 孙嗣国[1] 于哲[1] 陈翔[1] 纪振钢[1] 

机构地区：[1]第四军医大学唐都医院全军骨肿瘤研究所,陕西西安710038

出　　处：《现代肿瘤医学》2008年第5期697-700,共4页Journal of Modern Oncology

摘　　要：目的:构建人存活素(survivin)基因的siRNA真核表达载体,探讨其对骨肉瘤细胞SOSP-9607凋亡的影响。方法:利用pSilencer3.0-H1neo,构建人存活素特异性的RNA干涉载体,转染SOSP-9607细胞,G418筛选稳定转染的细胞系。western blot检测survivin蛋白水平变化。透射电镜等形态学观察、AnnexinV法、Hoechst染色检测细胞凋亡。结果:成功构建了人存活素基因siRNA真核表达载体PS。获得了稳定转染的细胞系。与正常SOSP-9607细胞、阴性对照细胞相比,SOSP-9607/PS生长曲线十分平缓,存在显著差异(P<0.01)。westernblot显示SOSP-9607/PS细胞中蛋白表达减少。SOSP-9607/PS凋亡率增加(P<0.01)。结论:特异性siRNA能够明显抑制survivin基因在SOSP-9607细胞中的表达并明显促进细胞凋亡,这为进一步研究survivin在SOSP-9607细胞中的生物学功能和作用机制奠定了实验基础。Objective： To explore the effect of siRNA on the expression of survivin in gene and leading apoptosis in osteosarcoma cell line SOSP-9607 using siRNA eukaryotic expression vector. Methods： According to survivin cD-NA coding sequence, the specific RNA interference （RNAi） fragments targeting survivin gene were designed and synthesized, which were cloned into pSilencer 3.0 -H1 neoplasmid vector, and the siRNA eukaryotic expression vector siRNA survivin targeting survivin gene was constructed. After the vector was constructed, SOSP-9607 cells were transfected with negative control vector or RNAi vectors and selected by G418. Expression of protein of surviving in the stable transfected cells was investigated by Western blot. The above cells were cultured, thus growth curve was drawn. These cells cultured on cover slips were observed through electronmicroscopy. Apoptosis analysis was finished by Annexin V and Hoechst staining. Results： The specific siRNA eukaryotic expression vector PS targeting survivin gene were constructed successfully. The stable transfectants containing negative control vector and PS were obtained. Expression of protein of survivin was inhibited significantly in SOSP - 9607/PS cells , whereas survivin gene expression levels were hardly changed in other groups. Otherwise, apoptosic rate was increased significantly in SOSP - 9607/PS cells than other two group （P 〈0. 01. Conclusion： survivin gene expression can be suppressed markedly by specific siRNA which induced apoptosis in SOSP- 9607 cells, the current results establish the experimental foundation for further studying the biological functions and its mechanisms of survivin in SOSP-9607 cells.

关 键 词：骨肉瘤 存活素 RNA干涉 

分 类 号：R738.1[医药卫生—肿瘤]