【摘要】 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。更多还原

【Abstract】 Objective To construct an adenoviral vector-mediated siRNA expression system to inhibit RhoA gene expression and induce the apoptosis of hepatocarcinoma cells together with TNF-α and to comprehensively analyze the function and role of RhoA in tumor cells.Methods Based on a previously constructed siRNA plasmid of RhoA,an adenoviral vector-mediated siRNA expression system was constructed to control the expression of RhoA in HepG2 cells.The level of RhoA expression was detected with Western blot and RT-PCR.The constructed adenovirus was used to infect HepG2 cells and was augmented with TNF-α.HepG2 cells were analyzed using MTT and TUNEL assays.Result siRNA mediation of RhoA via an adenovirus vector inhibited the expression of RhoA in hepatocarcinoma cells according to the results of Western blot.The rate of inhibition was 76.48%,and semi-quantitative RT-RCR showed that mRNA transcription of the RhoA gene was reduced by nearly 74.46%.MTT results revealed no significant differences in the OD value for control cells(Ad-U6-control) and cells with the adenovirus vector(Ad-siRNA-RhoA) according to SPSS10.0 software.TUNEL results indicated that adenovirus-mediated siRNA together with TNF-α can induce the apoptosis of HepG2 cells.Conclusion An adenoviral vector-mediated siRNA expression system to suppress RhoA was successfully constructed and augmented with TNF-α to induce the apoptosis of HepG2 cells.This method should help with basic research on the function of tumor genes and gene therapies for cancer.更多还原