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目的:构建针对人环氧合酶-2(COX-2)基因编码区的短发夹状RNA(shRNA)真核表达载体质粒,观察其在不同时间点对人不同肝癌细胞株COX-2表达的影响.方法:以人COX-2mRNA编码区作为RNA干扰靶点,构建shRNA真核表达载体质粒WBH1和WBH2,应用阳离子脂质体分别转染人肝癌细胞株HepG2和Bel7402,利用逆转录聚合酶链反应和Westernblot法分别观察两株细胞转染后24,48,72,和96hCOX-2mRNA和蛋白的表达变化,检测抑制效果.结果:质粒在HepG2细胞和Bel7402细胞中的转染率分别约为60%和54%.WBH1导入细胞24,48,72和96h后,逆转录聚合酶链反应检测COX-2mRNA表达抑制率,HepG2细胞分别为18.5%,88.6%,52.8%和42.4%(P<0.01).Bel7402细胞分别为9%,45.1%,70.1%和56.3%(P<0.01).Westernblot法检测蛋白表达抑制率,HepG2细胞分别为10.3%,80.5%,45.3%和39.0%(P<0.01);Bel7402细胞分别为8.3%,40.2%,66.4%和35.6%(P<0.01).质粒WBH2对COX-2的表达无影响(P>0.05).结论:针对人COX-2的shRNA能高效特异的抑制不同肝癌细胞株的COX-2表达.HepG2细胞和Bel7402细胞分别以48h和72h抑制效果最明显.56.3%(P<0.01).Westernblot法检测蛋白表达抑制率,HepG2细胞分别为10.3%,80.5%,45.3%和39.0%(P<0.01);Bel7402细胞分别为8.3%,40.2%,66.4%和35.6%(P<0.01).质粒WBH2对COX-2的表达无影响(P>0.05).结论:针对人COX-2的shRNA能高效特异的抑制不同肝癌细胞株的COX-2表达.HepG2细胞和Bel7402细胞分别以48h和72h抑制效果最明显.
OBJECTIVE: To construct a short hairpin RNA (shRNA) eukaryotic expression plasmid targeting human cyclooxygenase-2 (COX-2) gene and observe the expression of COX-2 at different time points in human hepatocellular carcinoma cell lines .Methods: Human COX-2mRNA coding region was used as target of RNA interference to construct shRNA eukaryotic expression plasmids WBH1 and WBH2. Human hepatocellular carcinoma cell lines HepG2 and Bel7402 were transfected with cationic liposomes respectively. Reverse transcriptase polymerase The changes of COX-2 mRNA and protein expression at 24, 48, 72 and 96 h after transfection were observed by chain reaction and Western blot respectively.Results: The transfection rates of plasmid in HepG2 cells and Bel7402 cells were about 60% and 54%, respectively.Western blotting showed that the inhibitory rate of COX-2 mRNA expression was 18.5%, 88.6%, 52.8% and 42.4% in HepG2 cells after transduced with WBH1 for 24,48,72 and 96 h, respectively 0.01) .Bel7402 cells were 9%, 45.1%, 70.1% and 56.3%, respectively (P <0.01) .The inhibitory rate of protein expression in HepG2 cells was 10.3%, 80.5%, 45.3% and 39.0% 0.01), and Bel7402 cells were 8.3%, 40.2%, 66.4% and 35.6%, respectively (P <0.01) .The plasmid WBH2 had no effect on COX-2 expression.Conclusion: (P <0.01) .Western Blot method was used to detect the expression of COX-2, the inhibitory rate of HepG2 cells and Bel7402 cells were 48h and 72h, respectively (10.3%, 80.5%, 45.3% and 39.0%, respectively) (P <0.01), and Bel7402 cells were 8.3%, 40.2%, 66.4% and 35.6% respectively.Plasma WBH2 had no effect on the expression of COX-2 (P> 0.05) .Conclusion: shRNA targeting human COX-2 can efficiently and specifically inhibit the expression of COX-2 in different hepatocellular carcinoma cell lines, and the inhibitory effect of HepG2 cells and Bel7402 cells at 48h and 72h respectively is the most obvious.