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目的探讨应用超声靶向破坏微泡(UTMD)技术介导shRNA抑制小鼠肝癌细胞株JNK1基因表达的能力。方法构建并筛选shRNA最佳表达载体。体外培养肝癌细胞Hca-F,共分为5组:A组为空白对照组;B组为shRNA质粒组;C组为脂质体组;D组为超声微泡+超声辐照组;E组为脂质体+超声微泡+超声辐照组。应用倒置荧光显微镜观察转染率;荧光定量PCR检测JNK1的mRNA水平;Western-Blot检测JNK1的蛋白质表达。结果获得了shRNA干扰效果最好的表达载体。各组转染率比较:E组均大于B、C、D组(P均<0.05);C、D组之间差异无统计学意义(P>0.05)。荧光定量PCR和Western-Blot检测各组JNK1mRNA和蛋白表达比较:E组的JNK1mRNA和蛋白表达水平均最低(P均<0.05)。结论脂质体转染法与UTMD技术结合可以提高小鼠肝癌细胞株JNK1shRNA的转染效率,并能够增强基因表达的抑制效果。
Objective To investigate the effect of UTMD on the expression of JNK1 in mouse hepatocellular carcinoma cell lines. Methods The shRNA expression vector was constructed and screened. Hca-F cells were cultured in vitro. The cells were divided into 5 groups: A group as blank control group, B group as shRNA plasmid group, C group as liposome group, D group as ultrasound microbubbles + ultrasound irradiation group, E group Liposomes + ultrasound microbubbles + ultrasound irradiation group. Transfection efficiency was observed by inverted fluorescent microscope. The mRNA level of JNK1 was detected by real-time PCR. The protein expression of JNK1 was detected by Western-Blot. The results obtained shRNA expression of the best interference vector. The transfection rates in each group were significantly higher in group E than in groups B, C and D (all P <0.05). There was no significant difference between groups C and D (P> 0.05). Compared with JNK1 mRNA and protein expression in each group, the mRNA and protein expression of JNK1 in E group were the lowest (all P <0.05). Conclusion The combination of lipofection and UTMD can enhance the transfection efficiency of JNK1 shRNA in mouse hepatocellular carcinoma cell line and enhance the inhibitory effect of gene expression.