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目的:构建活化转录因子2(ATF-2)基因RNA干扰的真核表达载体,观察其对HepG2肝癌细胞株增殖及凋亡的影响。方法:根据ATF-2 mRNA序列,合成两条寡聚DNA片段,退火后克隆入质粒载体PBA-siU6,DNA测序鉴定重组质粒PBA-siATF-2。脂质体介导质粒转染HepG2细胞。Westernblot法检测ATF-2蛋白表达;MTT方法检测细胞增殖率,流式细胞术(FCM)检测细胞凋亡率。结果:ATF-2基因RNA干扰载体经测序分析证实插入64 bp序列正确无误;Westernblot法显示干扰组细胞内ATF-2蛋白表达量明显降低;ATF-2基因的下调导致HepG2细胞增殖阻滞和凋亡发生。结论:成功构建了ATF-2基因RNAi真核表达载体,下调HepG2细胞ATF-2基因表达,抑制细胞增殖,诱导细胞凋亡。
OBJECTIVE: To construct an eukaryotic expression vector for RNAi of activated transcription factor 2 (ATF-2) gene and observe its effect on proliferation and apoptosis of HepG2 hepatocellular carcinoma cell line. Methods: According to the sequence of ATF-2 mRNA, two oligo DNA fragments were synthesized and annealed and then cloned into the plasmid vector PBA-siU6. DNA sequencing identified the recombinant plasmid PBA-siATF-2. Liposome-mediated plasmids transfected HepG2 cells. The expression of ATF-2 protein was detected by Western blotting. The cell proliferation rate was detected by MTT assay and the apoptosis rate by flow cytometry (FCM). Results: ATF-2 gene RNA interference vector was confirmed by sequencing analysis, insert the 64 bp sequence correct; Western blot showed that the expression of ATF-2 protein in the interference group decreased significantly; ATF-2 gene down-regulation led to HepG2 cell proliferation and apoptosis Death happened. CONCLUSION: The eukaryotic expression vector of ATF-2 gene RNAi was successfully constructed and the expression of ATF-2 gene was down-regulated in HepG2 cells, which inhibited cell proliferation and induced apoptosis.