热疗增强mTOR靶向基因治疗体外杀伤肝癌细胞的实验研究

Enhanced killing effect of hyperthermia of mTOR-targeted gene therapy on SMMC-7721 in vitro

目的:观察热疗对哺乳动物雷帕霉素靶蛋白(mTOR)靶向基因治疗在人肝癌细胞SMMC-7721中体外杀伤作用的影响。方法:观察不同温度热疗处理后SMMC-7721的形态学变化,并采用CCK-8试剂检测细胞增殖确定合适热疗温度,以该温度与转染pEGFP-C1-mTOR反义质粒相结合,设置联合组、转染组及对照组,CCK-8试剂检测处理后各组细胞增殖变化;流式细胞仪分析凋亡;逆转录PCR(RT-PCR)和蛋白免疫印记法检测mTOR基因表达。结果:热疗后细胞皱缩、变圆,胞浆空泡化,坏死细胞比例增加。CCK-8检测示温度越高,热疗对细胞增殖抑制作用越明显,42℃热疗后抑制率达(29.7±1.7)%。联合处理后细胞增殖能力降低,流式细胞仪分析示细胞凋亡比例增加,差异有统计学意义(P<0.05)。转染后mTOR基因表达在mR-NA水平和蛋白水平均受到抑制,而联合处理后表达水平进一步下降。结论:热疗可以增强mTOR靶向基因治疗对SMMC-7721细胞的体外杀伤作用。

Objective ： To investigate the enhanced killing effect of hyperthermia of mammalian target of rapa- mycin （mTOR） -targeted gene therapy on human hepatocellular carcinoma cell line SMMC-7721 in vitro. Methods： The morphological change of SMMC-7721 was observed after hyperthermia at different temperatures, thus the optimal temperature was determined with assistance of CCK-8 assay. Optimal hyperthermia was executed after trans fection of antisense plasmid pEGFP-CI-mTOR. Cell proliferation and apoptosis were evaluated by using CCK-8 as say and flow cytometry, respectively. The expression of mTOR mRNA and protein were analyzed by RT-PCR and Western blot. Results： After hyperthermia, cell shrinkage, rounded and blebbing, and also the proportion of necrotic cell were increased. According to CCK-8 assay, the inhibition effect was in consistent with the elevation of temperature, and reached 29.7± 1.7% at 42℃. After combined treatment, cell proliferation was significantly re- duced and with increased apoptosis （P 〈 0.05 ）. The expression of mTOR mRNA and protein were reduced after transfection, and more prominent combined treatment group. Conclusion： Hyperthermia enhances the killing effect of mTOR-targeted gene therapy on human hepatocellular carcinoma cell line SMMC-7721 in vitro.