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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示,与各对照组相比,survivin反义寡核苷酸和bcl-2反义寡核苷酸均可诱导细胞凋亡,凋亡率分别为43.6%和30.7%,两者联用凋亡率提高到58.6%(P<0.01)。结论①survivin反义寡核苷酸能抑制肺癌细胞株survivin基因表达,并诱导细胞凋亡和抑制生长。②survivin反义寡核苷酸联合bcl-2反义寡核苷酸具有显著协同抗癌作用。
OBJECTIVE: To investigate the effect of antisense oligonucleotide targeting survivin gene on human lung cancer cell lines and to explore the role of survivin antisense oligonucleotide in antitumor. Methods ① The experiment was performed in immunology laboratory of Bengbu Medical College from August 2002 to April 2003. The NCI-H446 cells in logarithmic growth phase were divided into six groups: blank control group, liposome 10m g / L group (only empty liposomes), NSODN500nm ol / L group (the three groups were control group), survivin Antisense oligodeoxynucleotide 500 nm ol / L group, bcl-2 antisense oligonucleotide 5 μm ol / L, survivin antisense oligonucleotide 500 nm ol / L + bcl-2 antisense oligonucleotide 5 μm ol / L group (transfected with different oligonucleotides under liposome-mediated, 48h after the cells were collected for the following test). ② The liposome-mediated antisense oligonucleotides of survivin were used to detect the expression of survivin mRNA in lung cancer cell lines NCI-H446 and SPC-A1 at 72h. Apoptosis Index = (quiescent / pre-DNA synthesis) percentage of the entire cell cycle; Proliferation index = (DNA replication phase + post-synthesis / mitosis) as a percentage of the entire cell cycle. ② Survivin antisense oligonucleotide alone or in combination with bcl-2 antisense oligonucleotide NCI-H446, the growth inhibition rate was measured by MTT method and calculated the two drug interaction index, Trypan Blue Anti-dye test to detect cell death rate. ③ measurement data were compared using variance analysis and q test. Results ① Both lung cancer cell lines expressed survivin gene. Survivin antisense oligonucleotides inhibited cell growth and inhibited cell apoptosis. Survivin m RNA was significantly down-regulated in A549 cells after antisense oligodeoxynucleotides (500 nmol / L) for 72 h RNA inhibition rates were 62.72% and 67.43% respectively. ② The results of MTT assay showed that both survivin antisense oligonucleotide and bcl-2 antisense oligonucleotide alone could inhibit the growth of NCI-H446 cells. Combined with survivin antisense oligonucleotide The cell growth inhibition rate of the nucleotide and bcl-2 antisense oligonucleotides was 64.9%, which was better than that of the bcl-2 antisense oligonucleotide (34.2%) (P <0.01) The action index is 0.708. Trypan blue exclusion assay showed that the cell death rate of survivin antisense oligonucleotide 500 nm ol / L + bcl-2 antisense oligonucleotide at 5 μm ol / L was 62.1%, higher than 31.4% And 41.4%. Apoptosis detected by flow cytometry showed that both survivin antisense oligonucleotide and bcl-2 antisense oligonucleotide induced apoptosis compared with the control group, the apoptosis rates were 43.6% and 30.7% respectively , The combined apoptosis rate increased to 58.6% (P <0.01). Conclusion ① Survivin antisense oligonucleotide can inhibit the expression of survivin gene in lung cancer cell lines and induce apoptosis and inhibit the growth of lung cancer cell lines. ② Survivin antisense oligonucleotides combined with bcl-2 antisense oligonucleotides have significant synergistic anti-cancer effects.