JWA基因参与细胞氧化应激的机制研究

目的　建立氧化应激模型 ,探讨新基因JWA参与细胞氧化应激的机制。方法　用H2 O2处理人乳腺癌细胞株 (MCF 7)和人胚胎肺纤维细胞株 (WI 38)两种细胞 ,观察细胞培养上清中丙二醛(MDA)和还原型谷胱甘肽 (GSH)的含量 ,用RT PCR半定量的方法及Westernblot蛋白质印迹法分别检测JWA在mRNA水平和蛋白质水平上的表达以及应激蛋白HSP2 7、HSP70、HSP90的表达。结果　MCF 7细胞培养上清中H2 O2 处理前MDA为 (0 .5 31± 0 .0 38)mmol/L ,处理后为 (0 .6 74± 0 .0 4 1)mmol/L ,差异有显著性 (P <0 .0 1) ;GSH在H2 O2 处理前为 (0 .0 5 3± 0 .0 0 2 )g/L ,处理后为 (0 .0 4 4±0 .0 0 2 )g/L ,差异有显著性 (P <0 .0 1)。WI 38细胞培养上清中H2 O2 处理前MDA的含量为 (0 .5 72±0 .0 35 )mmol/L ,处理后为 (0 .6 83± 0 .0 2 8)mmol/L ,差异有显著性 (P <0 .0 1) ;H2 O2 处理前GSH含量为(0 .0 5 8± 0 .0 0 2 )g/L ,处理后为 (0 .0 5 0± 0 .0 0 2 )g/L ,差异有显著性 (P <0 .0 1)。经H2 O2 孵育不同时间后 ,JWAmRNA在MCF 7细胞中明显下降 ,6h下降 6 8.4 % ,在WI 38细胞中则变化不明显 ;JWA蛋白和HSP2 7在两种细胞中的表达水平都有明显增加 ,但增加的程度不同。结论　JWA基因参与细胞氧化应激且在不同类型的细胞中

The mechanism of JWA gene involved in oxidative stress of cells

OBJECTIVE: To study the mechanism of gene JWA involved in oxidative stress under hydrogen peroxide (H(2)O(2)) exposure. METHODS: Both MCF-7 (human breast cancer cell line) and WI-38 (human embryo lung fibroblast cell line) cells were treated with 1 mmol/L of H(2)O(2) with or without pre-incubation of taurine (tau). Malondialdehyde (MDA) and glutathione (GSH) contents in supernatant of cell culture were measured; RT-PCR and Western blotting were carried out for evaluation of the expressions of JWA mRNA and protein respectively. Heat shock proteins (HSP27, HSP70 and HSP90) were also analyzed. RESULTS: The contents of MDA before and after H(2)O(2) treatment in MCF-7 cells were (0.531 +/- 0.038), (0.674 +/- 0.410) mmol/L respectively, (P < 0.01), while those in WI-38 cells were (0.572 +/- 0.035), (0.683 +/- 0.028) mmol/L respectively, (P < 0.01). The contents of GSH before and after H(2)O(2) treatment in MCF-7 cells were (0.053 +/- 0.002), (0.044 +/- 0.002) g/L respectively, (P < 0.01), while those in WI-38 cells were (0.058 +/- 0.002), (0.050 +/- 0.002) g/L respectively, (P < 0.01). The expression of JWA mRNA was down regulated, at 6 h it decreased by 68.4%, while in WI-38 cells no obvious change found. JWA protein and HSP27 showed markedly increased after H(2)O(2) treatment in both cells but not in similar extent. CONCLUSION: Oxidative stress signal pathways of JWA gene varied between cancer and non-cancer cell lines; JWA protein may have a similar function as HSP27 and act as an important signal molecule in H(2)O(2) induced cell injury.