超声靶向破坏微泡技术介导小鼠肝癌细胞株JNK1基因表达、细胞迁移和侵袭抑制

Inhibition of expression of JNK1 in mouse hepatocellular carcinoma cell lines and cell migration and invasion mediated by ultrasound-targeted microbubble destruction

目的:探讨应用超声靶向破坏微泡(UTMD)技术介导小鼠肝癌细胞株JNK1基因的表达、细胞迁移和侵袭抑制的作用,阐明其作用机制。方法:构建并筛选RNA干扰效果最好的短发夹RNA(shRNA)。将小鼠肝癌细胞株Hca-F分为正常Hca-F细胞组、shRNA质粒组、脂质体组、超声微泡结合超声辐照组及脂质体结合超声微泡加超声辐照组。采用倒置荧光显微镜观察各组细胞转染率,荧光定量PCR和Western blotting法检测JNK1基因mRNA和蛋白表达水平,CCK-8法检测各组细胞的细胞活性,应用Transwell实验检测各组细胞的体外迁移能力。结果:脂质体结合超声微泡加超声辐照组细胞转染率高于shRNA质粒组、脂质体组和超声微泡结合超声辐照组(均P<0.05),脂质体组和超声微泡结合超声辐照组比较差异无统计学意义(P>0.05)。脂质体结合超声微泡加超声辐照组JNK1mRNA和蛋白表达水平低于其他各组(P<0.05);脂质体结合超声微泡加超声辐照组细胞活性和平均穿膜细胞数均低于其他各组(P<0.05)。结论:UTMD技术结合脂质体转染法可以提高小鼠肝癌细胞株JNK1shRNA的转染效率,增强其对基因表达、细胞活力、迁移和侵袭能力的抑制。

Objective To explore the inhibition of the expression of JNK1 in mouse hepatocellular carcinoma cell lines and cell migration and invasion mediated by ultrasound-targeted microbubble destruction（UTMD）,and to clarify its mechanism.Methods The best short hairpin RNA（shRNA）vector was established and screened.The hepatocellular cell line Hca-F were cultured in vitro and divided into normal Hca-F cells group,shRNA plasmid group,and lipofection group,ultrasound microbubbles combined with ultrasound irradiation group,lipofection combined with ultrasound microbubbles and ultrasound irradiation group.The transfection rates were observed by inverted flurescence microscope.The expression levels of JNK1 mRNA and protein were detected by fluorescence quantitative PCR and Western blotting method.The activities of the cells in varions groups were detected by CCK-8method.The abilities of migration in vitro of the cells in various groups were detected by Transwell assay.Results The transfection rate of the cells in lipofection combined with ultrasound microbubbles and ultrasound irradiation group was higher than those in shRNA plasmid group,lipofection group and ultrasound microbubbles combined with ultrasound irradiation group（all P〈0.05）;there was no signifant difference between lipofection group and ultrasound microbubbles combined with ultrasound irradiation group（P〉0.05）.The expression levels of JNK1 mRNA and protein in lipofection combined with ultrasound microbubbles and ultrasound irradiation group were lower than those in other groups（all P〈0.05）.The cell activity and the average amount of the cells passed the membrane in lipofection combined with ultrasound microbubbles and ultrasound irradiation group were lower than those in other groups（all P 〈0.05）.Conclusion Combination of lipofection and UTMD in mouse hepatocellular cell lines can improve the transfection rate of JNK1 shRNA,therefore enhance the inhibitory effects on gene expression,the cell activity,migration and invasion.