沉默survivin基因介导口腔表皮癌细胞KB及KBv200凋亡的比较研究  

Study on Apoptosis of Oral Squamous Carcinoma Cell line KB and KBv200 induced by Survivin RNAi

作　　者：闫琳[1] 陈伟强[2] 梁永钜[1] 戴春岭[1] 王晓红[1] 石智[1] 陈黎明[1] 符立梧[1] 

机构地区：[1]华南肿瘤学国家重点实验室 [2]中山市陈星海医院,广东中山528400

出　　处：《癌症》2006年第8期933-940,共8页Chinese Journal of Cancer

基　　金：教育部博士点专项科研基金(No.20050558062);广东省医学科学基金(No.A2004785)~~

摘　　要：背景与目的:Survivin是IAPs家族的重要成员之一,研究表明可结合凋亡途径中的Caspase-3、Caspase-7、Caspase-9抑制其活性,引起肿瘤细胞的抗凋亡作用,尤其使肿瘤细胞耐受化疗药物诱导的凋亡,与化疗耐药密切相关。survivin在人口腔表皮癌细胞KB及其耐药株KBv200均有表达,本实验拟通过RNAi方法沉默survivin基因比较研究它介导KB和KBv200细胞的凋亡作用。方法:RT-PCR法检测细胞中survivinmRNA水平,流式细胞仪检测细胞中survivin蛋白水平,Hoechst33258荧光染色法观察细胞形态,PI染色结合流式细胞仪法检测细胞凋亡率,以及Caspase-3活性测定试剂盒检测Caspase-3的活性,MTT法测定肿瘤细胞生长情况。结果:KBv200细胞中survivinmRNA和蛋白表达量较高。转染mu6/survivin质粒后48h,KB和KBv200细胞中survivinmRNA表达抑制率分别为(61.98±8.12)%和(59.29±6.32)%,蛋白表达抑制率分别为(44.25±5.26)%和(38.76±4.31)%。转染mu6/survivin质粒后24h、48h、72h,流式细胞仪结果显示KB和KBv200细胞凋亡都明显增加,均在48h凋亡达到高峰,以KB凋亡率较高,并且Caspase-3活性也都明显增加,均在48h达到最高。同时KB细胞生长抑制率分别为(33.04±2.17)%、(56.25±3.32)%和(58.26±5.15)%,KBv200细胞生长抑制率则分别为(35.23±3.43)%、(44.90±4.12)%和(44.19±4.37)%。结论:siRNA方法能够有效沉默survivin基因,不仅能诱导KB细胞凋亡,而且能介导耐药株KBv200细胞凋亡。BACKGROUND ＆ OBJECTIVE： Survivin, a member of lAPs （inhibition of apoptosis proteins） family, has been revealed by many studies to inhibit the activation of caspase-3, caspase-7 and caspase-9, and thus increase the anti-apoptotic effect of cancer cells, which subsequently makes the cells to become resistant to chemotherapy in cancer treatment. Survivin is expressed in human oral squamous carcinoma cell line KB and its mutipledrug resistant （MDR） cell line KBv200. This study was to compare the apoptosis induced by silencing survivin gene between these two squamous cell lines. METHODS： Mu6/survivin plasmid which expressed shRNA against survivin was transfected into both KB and KBv200 cells to inhibit the expression of survivin gene. Survivin mRNA and protein expression was detected by semi-quantitative RT-PCR and flow cytometry （FCM）, respectively. Cell apoptotic rate was measured by flow cytometry after PI staining, and apoptotic morphology of cells was observed under a fluorescent microscope after Hoechst33258 staining. Activation of caspase-3 was measured by colorimetric assay. MTT was used to evaluate the growth inhibition of tumor cells. RESULTS： Expression of survivin, which was higher in KBv200 at mRNA and protein level, was detected in both KB and KBv200. After 48 h transfection with mu6/survivin plasmid, the expression level of survivin mRNA in KB and KBv200 was reduced by （61.98±8.12）% and （59.29±6.32）%; and that of survivin protein was decreased by （44.25±5.26）% and （38.76±4.31）% compared with the control group. Moreover, KB and KBv200 cells exhibited typical apoptotic cell morphology by Hoechst 33258 staining. Apoptosis, which was higher in KB cells, was increased significantly at 24, 48, 72 h after the transfection of mu6/surviving in KB and KBv200 cells, and reached the peak at 48h. Similarly, activation of caspase-3 was elevated significantly, and reached the peak at 48h. MIT assay revealed the inhibitory rates were （33.04±2.16）%, （56.25±3.32）% and �

关 键 词：RNA干扰 survivin凋亡 口腔上皮癌细胞 

分 类 号：R739.8[医药卫生—肿瘤]