摘要
目的:观察survivin反义寡核苷酸(antisense oligonucleotide,AS ODN)诱导肝癌细胞凋亡的可能途径。方法:培养survivin表达阳性的肝癌细胞株SMMC-7721。设计合成特异性靶向survivin的反义寡核苷酸。将SMMC-7721细胞接种于6孔培养板内,分为6组:①空白对照组;②脂质体转染对照组;③正义链转染对照组;④200nmol/L AS ODN转染组;⑤400nmol/L AS ODN转染组;⑥600nmol/L AS ODN转染组。作用20小时后收获各组细胞,并进行后续实验:①倒置显微镜下观察细胞形态变化;②western blot法检测各组细胞survivin蛋白的表达情况;③流式细胞术检测细胞增殖和凋亡指数;④比色法检测各组细胞caspase-3的活性变化。结果:①AS ODN转染组细胞survivin表达减弱,以600nmol/L转染组最为明显,各对照组survivin蛋白表达无明显改变;②AS ODN转染组细胞形态出现变圆、折光增强、漂浮、细胞碎片形成等变化,而各对照组细胞生长良好;③各AS ODN转染组细胞凋亡指数明显高于各对照组(P<0.05),以600nmol/L转染组最为明显,而各对照组间差异无显著性意义(P>0.05);④各AS ODN转染组细胞增殖指数明显低于各对照组(P<0.05),以600nmol/L转染组最为明显,而各对照组间差异无显著性意义;⑤在各AS ODN转染组中,可见到不同程度的caspase-3的活化,而对照组细胞内无明显的变化。结论:survivin AS ODN转染能下调survivin蛋白表达,诱导肝癌细胞凋亡,抑制细胞增殖,其作用途径可能涉及抑制survivin表达,诱导caspase-3活化,启动凋亡信号传导。
Objective： To investigate the path of apoptosis induced by antisense oligonucleotide （AS ODN） targeting survivin on hepatocellular cell. Methods： Positive expression of survivin had been confirmed in SMMC-7721 cell line. Antisense oligonucleotide targeting survivin was designed and composed. Twenty-four hours before transfection, SMMC-7721 cell were cultured in 6-well culture dish. Cultured cells were divided into 6 groups： ①control group; ②lipofectin group; ③ODN group; ④200nmol/L AS ODN group; ⑤400nmol/L AS ODN group; ⑥600nmol/L AS ODN group. After transfection for 20 hours, cultured cells were harvested to continue next tests. ①Cell form was observed with invert microscope; ②Survivin protein expression was detected by western blot; ③Apoptotic index （AI） and proliferative index （PI） were examined by flow cytometry; ④Activity of caspase-3 was detected by western blot. Results： ①Expression of survivin in AS ODN groups were significantly decreased than that of the control, lipofectin and ODN group; ②Abnormal form of cells was observed in AS ODN transfected groups ; ③The PI of AS ODN groups were significantly higher than that of other groups ; ④The PI of AS ODN groups were significantly lower than that of other groups; ⑤Activity of caspase-3 was increased in AS ODN groups but not in other groups. Conclusion： Antisense oligonucleotide targeting survivin could decrease the expression of survivin, induce hepatomacellular apoptosis and inhibit cell proliferation.
出处
《中西医结合肝病杂志》
CAS
2007年第3期151-153,共3页
Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases
