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目的建立RhoA基因的腺病毒siRNA系统,并联合TNF-α诱导肝癌细胞凋亡,分析RhoA在肿瘤细胞中的功能和作用。方法用已构建的RhoA干扰质粒构建RhoA的腺病毒siRNA系统,筛选重组病毒并感染HepG2细胞,应用Western blot和RT-PCR检测RhoA蛋白表达和基因表达水平。感染Ad-siRNA-RhoA腺病毒的肝癌细胞用TNF-α诱导后进行MTT以及细胞内DNA片段化检测。结果成功构建RhoA基因的siRNA腺病毒系统。用重组病毒感染肝癌细胞,RhoA蛋白表达抑制率为76.48%;RhoA基因的mRNA转录水平降低74.46%。MTT检测显示,感染腺病毒Ad-U6-control对照组以及感染腺病毒Ad-siRNA-RhoA实验组的细胞A值差异无统计学意义(F=5.41,P>0.01),即利用siRNA抑制肝癌细胞中RhoA表达,肿瘤细胞凋亡不明显。TUNEL检测显示,RhoA腺病毒siRNA联合TNF-α致肿瘤细胞凋亡作用显著。结论构建的腺病毒siRNA载体系统能抑制目的基因RhoA的表达,联合TNF-α能抑制肿瘤细胞生长和增殖并诱导肿瘤细胞凋亡,为肿瘤基因功能的基础研究和肿瘤基因治疗打下了实验基础。
Objective To establish a adenovirus siRNA system targeting RhoA gene and induce apoptosis of hepatoma cells in combination with TNF-α, and to analyze the function and function of RhoA in tumor cells. Methods The constructed RhoA siRNA plasmid was used to construct RhoA adenovirus siRNA system. The recombinant virus was screened and infected into HepG2 cells. The protein expression and gene expression of RhoA were detected by Western blot and RT-PCR. Liver cancer cells infected with Ad-siRNA-RhoA adenovirus were induced by TNF-α, and MTT and intracellular DNA fragmentation were detected. Results The siRNA adenovirus system of RhoA gene was successfully constructed. Infection of hepatoma cells with recombinant virus, RhoA protein expression inhibition rate was 76.48%; RhoA gene mRNA transcription level decreased 74.46%. The results of MTT assay showed that there was no significant difference of A value between Ad-U6-control group and Ad-siRNA-RhoA group (F = 5.41, P> 0.01) RhoA expression, tumor cell apoptosis was not obvious. TUNEL assay showed that RhoA adenovirus siRNA combined with TNF-α induced tumor cell apoptosis significantly. CONCLUSION: The constructed adenovirus siRNA vector can inhibit the expression of RhoA gene. Combined with TNF-α, it can inhibit the growth and proliferation of tumor cells and induce the apoptosis of tumor cells, laying a foundation for the basic research of tumor gene function and tumor gene therapy.