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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 investigate the role of UTMD in the inhibition of JNK1 gene expression, cell migration and invasion in mouse hepatocellular carcinoma cell lines and to elucidate its mechanism of action. Methods: We constructed and screened short hairpin RNA (shRNA) with the best RNA interference. Hca-F cells were divided into normal Hca-F cell group, shRNA plasmid group, liposome group, ultrasound microbubble combined with ultrasound irradiation group and liposome combined with ultrasound microbubble plus ultrasound irradiation group. The transfection rate of JNK1 mRNA and protein were detected by inverted fluorescence microscope. The expression of JNK1 mRNA and protein was detected by real-time quantitative PCR and Western blotting. The cell viability was measured by CCK-8 assay. Transwell assay was used to detect the in vitro migration ability. Results: The transfection efficiency of liposome combined with ultrasound microbubbles plus ultrasound irradiation was higher than that of shRNA plasmid group, liposome group and ultrasound microbubbles combined ultrasound irradiation group (all P <0.05), liposome group and ultrasound There was no significant difference between the microbubbles combined ultrasound irradiation group (P> 0.05). Compared with the other groups, the expression of JNK1mRNA and protein in liposome-combined ultrasound microbubble plus ultrasound irradiation group was lower than that in other groups (P <0.05). The cell viability and average number of transmembrane cells in liposome combined with ultrasound microbubble plus ultrasound irradiation were lower In other groups (P <0.05). CONCLUSION: UTMD combined with lipofection can increase transfection efficiency of JNK1 shRNA in mouse hepatocellular carcinoma cell line and increase its inhibitory effect on gene expression, cell viability, migration and invasion.