摘要
目的构建胰腺癌细胞生存蛋白(survivin)表达的RNA干扰(RNAi)抑制载体,并研究其对survivin表达的抑制作用。方法用免疫荧光技术和RT-PCR检测胰腺癌细胞PANC-1中survivin及其mRNA的表达,并把survivin基因克隆到T载体进行测序;构建针对survivin缺失碱基基因和survivin基因的RNAi载体si-svv-1和si-svv-2,用脂质体转染胰腺癌细胞PANC-1后,用RT-PCR、DNA梯状电泳及流式细胞术分析比较2种干扰载体对survivinmRNA表达的抑制情况和检测细胞凋亡。结果survivin在胰腺癌细胞PANC-1中高表达;si-svv-2对survivinmRNA的表达抑制率达(72.43±8.04)%,而si-svv-1为0;si-svv-2能诱导胰腺癌细胞PANC-1的凋亡,72h细胞凋亡率达(12.36±1.44)%。结论本研究成功建立胰腺癌细胞survivin表达RNAi抑制载体,该载体可特异有效地抑制胰腺癌细胞PANC-1survivin的表达并诱导胰腺癌细胞PANC-1凋亡。
Objective To investigate the inhibitory effects of RNA interference （RNAi） expression vector on the expression of survivin in pancreatic cancer cell PANC-1. Methods The protein and mRNA expressions of survivin were examined with immunofluoreseence and RT-PCR. The survivin gene was cloned into the T-vector and sequenced. The RNAi expression vectors targeting survivin, named si-svv-1 and si-svv-2 respectively according to whether they harbored a mutation or no mutation, were constructed and transfected into PANC 1 cells with liposome. The expression of survivin mRNA was detected with RT-PCR. Apoptosis of PANC-1 ceils was analyzed with DNA ladder and FACS. Results There was a high degree expression of survivin in PANC-1 cells. The expression of survivin was not inhibited by RNAi expression vectors si-svv-1, but inhibited about （72.43±8.04） % by si-svv-2 and the apoptosis rate of PANC 1 cells increased to （12.36±1.44） % after 72 h. Conclusion The RNAi expression vector can effectively inhibit the expression of survivin in pancreatic cancer cell PANC-1 cells and induce the apoptosis in PANC-1 cells.
出处
《中国普外基础与临床杂志》
CAS
2008年第11期829-834,共6页
Chinese Journal of Bases and Clinics In General Surgery
关键词
RNA干扰
胰腺癌
生存蛋白
凋亡
RNA interference Pancreatic cancer Survivin Apoptosis
