Survivin反义寡核苷酸诱导肝癌细胞凋亡及细胞结构的变化

目的采用反义寡核苷酸封闭肝癌细胞中Survivin基因的表达，研究其诱导细胞凋亡过程中对细胞超微结构与细胞骨架的作用及其机理。方法采用脂质体介导Survivin反义寡核苷酸转染人肝癌细胞株SMMC-7721细胞，Western—blot及原位杂交方法检测Survivin蛋白及mRNA表达，流式细胞仪检测细胞凋亡比率，透射电子显微镜观察细胞超微结构变化，激光共聚焦显微镜观察细胞骨架微丝系统变化，激酶活性检测方法测定细胞内Caspase-3活性变化。结果脂质体介导Survivin反义寡核苷酸转染肝癌细胞后Survivin蛋白及mRNA表达分别由69．59及75．60降低至10．71及22．90，Caspase-3活性由0．0153升高至0．0992，同时细胞结构呈典型凋亡改变，细胞内微丝形态结构破坏，细胞凋亡比率由0．7％增加至31．4％。结论脂质体介导转染Survivin反义寡核苷酸可以有效降低细胞内Survivin基因的表达，并激活Caspase-3。活化的Caspase-3可以切割破坏细胞内骨架微丝系统的结构，进而诱导细胞凋亡。

Changes of hepatocellular carcinoma cell structures during apoptosis induced by Survivin antisense oligonucleotide

ObjectivesTo investigate the changes of hepatocellular carcinoma cell line SMMC-7721 in cell ultrastructure and skeleton during apoptosis induced by Survivin ASODN.MethodsSurvivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent. The changes of Survivin protein and mRNA expression after transfection were assessed by Western blot and in situ hybridization. The apoptotic rate was detected by flow cytometer. Cell ultrastructure and skeleton were observed by electron microscope and confocal microscope. Caspase-3 was assessed by kinase activity assess method. Results The expression of Survivin protein and mRNA decreased from 69.59 and 75.6 to 10.71 and 22.9 respectively. The cell ultrastructure showed apoptotic changes. Cell skeleton microfilament system changed after Survivin ASODN transfection. The average intensity of microfilament system fluorescent decreased from 190 to 64. The activity of Caspase-3 increased from 0.015 3 to 0.099 2.ConclusionsTransfection of ASODN targeted to Survivin mRNA by DOTAP liposomal transfection reagent down-regulated the expression of Survivin protein and mRNA in SMMC 7721 cell line and activate Caspase-3, which in turns induced cell apoptosis.