survivin抗肺癌细胞凋亡的分子机制  

Molecular mechanism of anfi-apoptotic action of survivin in NCI-H446 lung cancer cells

作　　者：陈余清[1] 李伟[1] 周继红[2] 李殿明[1] 夏雪梅[1] 黄礼年[1] 李柏青[3] 

机构地区：[1]蚌埠医学院第一附属医院呼吸科安徽省卫生厅临床重点专科,233004 [2]蚌埠医学院生物化学与分子生物学教研室 [3]免疫学教研室安徽省教育厅重点实验室

出　　处：《中华肿瘤杂志》2006年第6期413-417,共5页Chinese Journal of Oncology

基　　金：安徽省自然科学基金(03043705);安徽省教育厅自然科学基金(2004kj283)

摘　　要：目的研究survivin反义寡核苷酸(ASODN)诱导肺癌细胞凋亡的作用,探讨survivin抗肺癌细胞凋亡的分子机制。方法在脂质体介导下,分别以100 mmol/L、300 mmol/L和500 mmol/L浓度的survivin ASODN,作用于肺癌细胞株NCI-H446 24 h、48 h和72 h后,用RT-PCR和Western blot检测survivin mRNA及蛋白表达,流式细胞仪(FCM)检测细胞凋亡；Rh123染色法检测细胞线粒体膜电位(△Ψm)变化；ELISA法检测胞浆细胞色素C(cyt C)浓度；比色法检测胞浆内天冬氨酸特异性半胱氨酸蛋白酶-9(caspase-9)、caspase-3的活性；Western blot检测caspase-8蛋白表达；加入环孢霉素A (CsA)后,以FCM检测细胞凋亡。结果与各对照组相比,survivin ASODN显著下调了survivin mRNA表达,且呈时间和浓度依赖性,其中以500 mmol/L作用72 h时效果最佳,抑制率达62．7%,其蛋白表达也显著下调。NCI-H446细胞的凋亡指数(AI)为48．35%,明显高于对照组(3．75%)、空脂质体组(3．41%)、无义寡核苷酸(NSODN)组(4．69%)、100和300 mmol/L ASODN组(19．85%和34．39%)；增殖指数(PI)为24．38%,明显低于上述各组(75．54%、73．12%、71．76%、51．03%和38．94%,P均＜0．01)。survivin ASODN导致细胞△Ψm逐渐下降,并相继引起cyt C的释放、caspase-9和caspase-3的激活,而caspase-8酶原无变化。CsA显著抑制了survivin ASODN诱导的细胞凋亡。结论survivin通过调控线粒体凋亡途径发挥抗肺癌细胞凋亡作用；survivin ASODN能够显著下调survivin mRNA及蛋白表达,诱导肺癌细胞凋亡,抑制细胞增殖。Objective To investigate cell apoptosis induced by survivin ASODN and clarify the precise mechanism of anti-apoptotic action of survivin. Methods Cells of lung cancer cell line NCI-H446 were treated with survivin ASODN at different concentrations. The changes of survivin mRNA and protein expression were assessed by RT-PCR and Western blot assay. The apoptosis index （ AI ） and proliferation index （PI） were determined by flow cytometry （FCM）. After 500 mmol/L survivin ASODN treatment, cells were stained with Rh123 to detect changes of mitochondrial membrane potential （△ψm） by FCM. The concentration of cytoplasmic cytochrome c （cyt-c） was continuously determined by ELISA. Relative activities of caspase-9 and caspase-3 were assessed by colorimetric assay. The expression of caspase-8 protein was measured by Western blot assay. The apoptotic rates of lung cancer cells induced by survivin ASODN with or without mitochondrial permeability transition pole （MPTP） inhibitor CsA treatment were assessed by FCM. Results Down-regulated survivin mRNA was shown to be in dose-dependent and timedependent manners. Its maximal effect was achieved at a concentration of 500 nmol/L for 72 h, at which mRNA was down-regulated by 62. 7% , the expression of survivin protein in NCI-H446 cells was also obviously decreased. After treatment with survivin ASODN at concentration of 500 mmol/L for 72 h, AI was 48.35%, higher than that of control, lipofectin, NSODN, survivin ASODN 100 mmol/L and 300 mmol/L groups （3.75% , 3.41%, 4.69%, 19.85% and 34.39%, respectively）. PI was 24.38%, lower than that of control, lipofectin, NSODN, survivin ASODN100 and 300 mmol/L groups （75.74%, 73. 12%, 71.76%, 51.03% and 38.94%, respectively）. △ψm was decreased in 9.54% of NCI-H446 cells treated with survivin ASODN for 3 h and 97.06% for 24 h. Following it, release of cyt-c from mitochondria to cytosol and activation of caspase-9 and caspase-3 increased significantly. The above mentioned indicators changed with a time-dependent

关 键 词：SURVIVIN 肺癌 细胞系 细胞凋亡 

分 类 号：R734.2[医药卫生—肿瘤]