RNAi抑制Smad4基因表达对猪卵巢颗粒细胞凋亡的影响  

Effects of Silencing Smad4 Gene by Small Interfering RNA on Apoptosis of Porcine Granulosa Cells

作　　者：张海燕[1] 王伟[1] 贺彧[1] 王莉[1] 田宽校[1] 宋晓光[1] 徐银学[1] 

机构地区：[1]南京农业大学动物科技学院,南京210095

出　　处：《中国细胞生物学学报》2010年第4期596-600,共5页Chinese Journal of Cell Biology

基　　金：国家高技术研究发展计划课题(No.2006AAl0Z136)资助项目~~

摘　　要：Smad蛋白家族是TGF-β/Smad信号通路中的重要成员,其中Smad4在该信号转导途径中起着关键作用。本研究利用RNAi技术沉默Smad4基因,探讨其对猪卵巢颗粒细胞凋亡的影响。设计并合成靶向Smad4的小分子干扰RNA,在脂质体的介导下转染猪卵巢颗粒细胞,荧光定量PCR检测Smad4 mRNA的表达情况;MTT(四甲基偶氮唑盐)比色法分析检测细胞活性;TUNEL(原位末端核苷酸标记法)及Annexin-V/PI双染流式细胞仪检测细胞凋亡情况;荧光定量PCR检测Bcl-2、Bax的mRNA表达水平。研究显示,Smad4-siRNA能有效抑制Smad4的mRNA表达(P<0.01),细胞活性由0.31减少到0.27,凋亡率由17.0%增加到22.1%,Bcl-2的mRNA表达显著下调。结果说明沉默Smad4基因可以促进猪卵巢颗粒细胞的凋亡,其机制可能与调控Bcl-2表达有关。Smad protein family is an important member of TGF-I3/Smad signal pathway in which Smad4 plays a key role. In this study, RNAi was used to explore the effect of silencing Smad4 gene expression on apoptosis of porcine Granulosa cells （PGC）. Chemically synthesized small interference RNA targeting to Smad4 was transfected into PGC using lipidosome. Smad4 mRNA expression level was assayed by real-time fluorescence quantitative PCR technology. Cytoactive, apoptotic and apoptofic index were analyzed by MTT colorimereic analysis, TUNEL and flow cytometry, respectively. Expression level of Bcl-2 and Bax mRNA was analyzed by real-time fluorescence quantitative PCR technology. Based on these studies, Smad4-siRNA effectively silenced the expression of Smad4 mRNA（P〈0.01）. The cytoactive decreased （0.31 to 0.27）, and the apoptotic rate increased（17.0% to 22.1%）, the expression of Bcl-2 mRNA was significantly decreased. These results suggested that silencing Smad4 gene can increase apoptosis of PGC, which possibly relate to regulation of the expression of Bcl-2.

关 键 词：猪卵巢颗粒细胞 SMAD4 RNA干扰 细胞凋亡 

分 类 号：Q343[生物学—遗传学]