凋亡抑制基因survivin反义寡核苷酸联合bcl-2反义寡核苷酸诱导肺癌细胞株凋亡的实验观察  

Apoptosis of lung cancer cell lines induced by apoptosis inhibiting gene survivin antisense oligonucleotide combined with bcl-2 antisense oligonucleotide

作　　者：夏雪梅[1] 陈余清[1] 蔡映云[2] 李殿明[1] 胡俊锋[1] 黄礼年[1] 

机构地区：[1]蚌埠医学院附属医院肺科,安徽省蚌埠市233004 [2]复旦大学附属中山医院肺科,上海市200032

出　　处：《中国临床康复》2005年第47期69-71,i0005,共4页Chinese Journal of Clinical Rehabilitation

基　　金：安徽省自然科学基金资助项目(03043705)~~

摘　　要：目的探讨凋亡抑制基因survivin反义寡核苷酸单用或联用bcl-2反义寡核苷酸对肺癌细胞株的作用,探讨其在抗癌方面的作用。方法①实验于2002-08/2003-04在蚌埠医学院免疫学实验室完成。将对数生长期NCI-H446细胞分为6组空白对照组,脂质体10m g/L组(仅加空脂质体),NSODN500nm ol/L组(该3组均为对照组),survivin反义寡核苷酸500nm ol/L组,bcl-2反义寡核苷酸5μm ol/L,survivin反义寡核苷酸500nm ol/L+bcl-2反义寡核苷酸5μm ol/L组(分别在脂质体介导下转染不同寡核苷酸,48h后收取细胞进行以下检测)。②在脂质体介导下,以survivin的反义寡核苷酸作用于肺癌细胞株NCI-H446和SPC-A1,于72h用反转录聚合酶链反应检测survivin mRNA表达。凋亡指数=(静止期/DNA合成前期)占整个细胞周期的百分比;增殖指数=(DNA复制期+合成后期/有丝分裂期)占整个细胞周期的百分比。②survivin反义寡核苷酸单独或联合bcl-2反义寡核苷酸作用NCI-H446后,用四甲基偶氮唑盐法检测细胞生长抑制率并计算两药相互作用指数,锥虫蓝拒染实验检测细胞死亡率。③计量资料差异性比较采用方差分析和q检验。结果①两种肺癌细胞株皆表达survivin基因。survivin反义寡核苷酸作用细胞株后,出现生长抑制和细胞凋亡,细胞survivin m RNA明显下调,反义寡核苷酸500nm ol/L作用72h后NCI-H446和SPC-A1细胞survivin m RNA抑制率分别达62.72%和67.43%。②四甲基偶氮唑盐法检测结果显示,survivin反义寡核苷酸和bcl-2反义寡核苷酸单独应用对NCI-H446细胞均有生长抑制作用;联合应用survivin反义寡核苷酸和bcl-2反义寡核苷酸的细胞生长抑制率为64.9%,优于单独应用(单用bcl-2反义寡核苷酸为34.2%)(P<0.01),两药相互作用指数为0.708。锥虫蓝拒染实验显示,survivin反义寡核苷酸500nm ol/L+bcl-2反义寡核苷酸5μm ol/L组细胞死亡率为62.1%,高于两药单用时的31.4%和41.4%。流式细胞仪检测凋亡显AIM： To investigate the synergic effect of survivin antisense oligonucleotide （ASODN） combined with bcl-2 ASODN on apoptosis in lung cancer cell lines, and probe into its anti-cancer effect. METHODS （1） The experiment was carried out in the immunological laboratory of Bengbu Medical College between August 2002 and April 2003. The NCI-H446 cells at logarithmic phase were divided into 6 groups： blank control group, lipusomes 10 mg/L group （only blank liposomes was added）, NSODN 500 nmol/L group （the first 3 groups were the control groups）, survivin ASOI）N 500 nmol/L group, bcl-2 ASODN 5 μmol/L group, survivin ASODN 500 nmol/L ＋bcl-2 ASODN 5 μmol/L group （different oligonucleotides were transfeeted under mediation of liposomes, and then the cells were collected for the following tests）. （2） Under medium of liposomes, the lung cancer cell lines, NCI-H446 and SPC-A1 were transferred with survivin ASODN, and the survivin mRNA expression was detected with reverse transcription-polymerase chain reaction （RTPCR） at 72 hours. Apoptosis index=（resting phase/DNA G1 phase）/whole cell cycle ×100%. Proliferation index=（DNA synthesis phase＋G2 phase/M phase/whole cell cycle ×100%. 3@ After NCI-H446 cell was treated with survivin ASODN alone or combined with bcl-2 ASODN, the cell growth inhibitory rate of NCI-H446 cell was detected with methyl-thiazol-tetrazolium （MTT） assay, and the interaction index of the two drugs was calculated, and the cell death rate was detected with trypan blue staining. （4） The analysis of variance and q test were applied in comaparing the difference of measurement data. RESULTS： （1） The surviving gone expression was observed in both lung cancer cell lines. After cell line was treated with survivin ASODN, the growth inhibition and apoptosis occurred, and the survivin mRNA was obviously down-regulated, and the survivin mRNA inhibitory rates of NCI- H446 and SPC-A1 cells after treated with ASODN for 72 hours were 62.72% and 67.43% respectively.

关 键 词：肺肿瘤 寡核苷酸类 反义 基因 bcl-2 细胞凋亡 

分 类 号：R734.2[医药卫生—肿瘤]