血管生成素-2基因沉默载体构建及其对肝癌细胞的作用

Construction of Lentiviral Vector of RNA Interference of Ang-2 Gene and Effect on hepG2 Cells in vitro

目的:构建血管生成素(ang)-2基因沉默载体,并研究其对肝癌hepG2细胞株的作用。方法:根据ang-2基因序列设计shRNA,并与载体pLVX-shRNA连接,转化感受态细胞,提取质粒测序鉴定。将慢病毒shRNA载体及其辅助包装原件载体质粒共转染293T细胞,在HEK293T细胞中标定其病毒滴度。用包装好的慢病毒以及慢病毒阴性对照感染靶细胞hepG2,感染成功后通过Western Blot方法评价干扰载体对靶基因的干扰效果,并采用MTT法检测其对靶细胞增殖率的影响。结果:酶切鉴定和测序结果证实成功构建了ang-2基因沉默载体。包装并浓缩慢病毒,滴度为1×10~8 TU/ml。将ang-2基因沉默载体感染hepG2细胞,倒置显微镜下可见绿色荧光。Western Blotting检测ang-2蛋白表达水平较对照组明显降低(P<0.05),而对hepG2细胞增殖率无明显影响。结论:成功构建了ang-2基因沉默载体并感染靶细胞hepG2,证实其对目的基因具有很好的沉默效果。

Objective. To construct lentiviral shRNA vector carrying ang-2 gene and to study the interference efficiency of ang-2 shRNA in hepG2 cell in vitro. Methods： shRNA was designed according to ang-2 gene sequence and connected to pLVX-shRNA vector. The connection product was switched to competent cells in bacteria. Plasmid DNA was extracted and sequenced. Lentiviral shRNA vector and auxiliary vector were transfected to 293T cells, and the lentiviral-rich supernatants was collected after promoting transfection, changing the solution and breeding. High titer of lentiviral was obtained and measured by condensing the supernatants. Lentiviral and NC-lentiviral were infected into hepG2 cells. Then jamming effect of lentiviral vector acting on target gene was evaluated by Western blot and cell proliferation rates by MTT. Results. Lentiviral shRNA vector carrying ang-2 gene was constructed successfully. The titer of concentrated virus was 1 × 10^8 TU/ml. The bepG2 cell line was infected with ang-2 gene silencing vector and green fluorescence could be observed. The expression of ang-2 protein in hepG2 cell line was inhibited, but cell proliferation rates were not influenced. Conclusion. shRNA vector carrying ang-2 gene was constructed successfully and confirmed to be effective for scilencing of target gene.