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目的构建T细胞转录因子(Tcf)反义RNA真核表达载体以阻断异常Wnt信号通路,探讨其对肝癌细胞生物学特性的影响。方法利用GeneJammer将反义基因转染人肝癌细胞系SMMC-7721,应用RT-PCR及Western blot方法分别检测转染前后细胞mRNA及蛋白表达差异,通过生长曲线、Transwell小室实验比较转染细胞生长增殖及运动侵袭能力,并进一步用流式细胞仪检测细胞凋亡及生长周期的改变。结果反义RNA转染降低了Tcf表达,减慢了肝癌细胞的生长速度并抑止其运动侵袭能力。转染反义RNA的肝癌细胞7721-pTas凋亡比例增加[(26.34±2.07)%],明显高于空载体转染细胞7721-vector[(6.53±1.02)%]和亲本SMMC-7721细胞[(4.33±0.68)%](P<0.001)。同时,处于G0-G1期的反义转染细胞比相应亲本SMMC-7721细胞和转染空载体的细胞7721-vector分别高20.24%和20.95%,而S期细胞比亲本细胞SMMC-7721和7721-vector细胞分别低11.8%和11.38%。结论反义TcfRNA转染可通过诱导细胞凋亡和阻止细胞周期的进程而抑制肝癌细胞的恶性增殖,提示选择性阻断异常Wnt信号通路有望成为肝癌基因治疗的新途径。
Objective To construct antisense RNA eukaryotic expression vector of T cell (Tcf) antisense RNA to block the abnormal Wnt signaling pathway and to investigate its effect on the biological characteristics of hepatoma cells. Methods Antisense gene was transfected into human hepatocellular carcinoma cell line SMMC-7721 with GeneJammer. The mRNA and protein expressions of the transfected cells were detected by RT-PCR and Western blot respectively. The growth curve and Transwell assay were used to compare the growth and proliferation of transfected cells And sports invasion ability, and further using flow cytometry to detect apoptosis and growth cycle changes. Results The antisense RNA transfection reduced the expression of Tcf, slowed the growth of hepatocellular carcinoma cells and inhibited the motility of invasion. The percentage of apoptotic cells in 7721-pTas cells transfected with antisense RNA increased significantly ([(26.34 ± 2.07)%], which was significantly higher than that in transfected cells 7721-vector [(6.53 ± 1.02)%] and parental SMMC-7721 cells (4.33 ± 0.68)%] (P <0.001). At the same time, the antisense transfected cells in G0-G1 phase were 20.24% and 20.95% higher than the corresponding parental SMMC-7721 cells and the 7721-vector transfected with empty vector, respectively. However, the S phase cells were significantly higher than the parental cells SMMC-7721 and 7721 -vector cells were 11.8% and 11.38% lower respectively. Conclusion Antisense TcfRNA transfection can inhibit the malignant proliferation of hepatocellular carcinoma cells by inducing apoptosis and preventing cell cycle progression, suggesting that selectively blocking the abnormal Wnt signaling pathway is expected to become a new way of gene therapy for hepatocellular carcinoma.