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以有义和反义人锰型超氧化物歧化酶 (MnSOD)、蛋白激酶B (PKB)cDNA分别转染人 772 1肝癌细胞系建立高、低表达MnSOD和PKB的模型 ,通过改变细胞内、外源性活性氧和抗氧化水平及PKB的活性 ,研究活性氧调节肝癌细胞生长过程中与PKB信号转导途径的关系。结果表明 ,低浓度外源性活性氧过氧化氢 (1～ 10μmol/L)应激及MnSOD低表达细胞中内源性活性氧水平升高 ,都可促进肝癌细胞增殖 ,而抗氧化剂丹参素 (4 0mg/L)干预及MnSOD高表达使细胞内源性活性氧水平相对下降 ,都可一定程度抑制细胞的生长。采用3 2 P参入法检测细胞PKB酶活性 ,发现细胞内、外源性活性氧均能激活PKB ,而采用抗氧化干预 ,能明显抑制PKB的酶活性。采用RT PCR法检测AP 1转录因子组成成员c fos和c jun基因的mRNA表达 ,发现与PKB活性呈正比。说明活性氧在特定细胞内氧化 /还原环境下可通过激活PKB途径传递信号、调控转录因子AP 1表达、促进肝癌细胞生长
The model of MnSOD and PKB was constructed by transfecting human 772-1 hepatoma cell line with sense and antisense human MnSOD and PKB cDNA respectively. Exogenous reactive oxygen species, antioxidative activity and PKB activity, and to study the relationship between reactive oxygen species (ROS) regulation of PKB signal transduction pathway and hepatoma cell growth. The results showed that low concentrations of exogenous hydrogen peroxide (1 ~ 10μmol / L) stress and MnSOD low expression of endogenous reactive oxygen species level increased, can promote the proliferation of liver cancer cells, and the antioxidant Danshensu 4 0 mg / L) and MnSOD overexpression decreased the levels of reactive oxygen species (ROS) in cells, which could inhibit the growth of cells to a certain extent. Using 3 2 P incorporation assay to detect the activity of PKB, we found that both intracellular and exogenous reactive oxygen species can activate PKB, and the antioxidant activity of PKB can be significantly inhibited by the antioxidant intervention. RT-PCR was used to detect the mRNA expression of c fos and c jun gene members of AP 1 transcription factor and found to be proportional to PKB activity. It shows that reactive oxygen species (ROS) can regulate the expression of AP 1 and promote the growth of hepatocarcinoma cells by activating the PKB pathway under specific intracellular oxidative / reductive conditions