DNA氧化损伤修复基因ERCC6对过氧化氢诱导的氧化损伤的晶状体上皮细胞增殖和凋亡的影响

目的　探讨DNA氧化损伤修复基因ERCC6对过氧化氢（H₂O₂）诱导的氧化损伤的晶状体上皮细胞增殖和凋亡的影响。方法　采用不同浓度H₂O₂处理SRA01/04细胞，构建氧化损伤模型。取氧化损伤的SRA01/04细胞分为H₂O₂组、H₂O₂+空白质粒转染组和H₂O₂+pcDNA3.1-ERCC6质粒转染组进行转染处理。采用实时荧光定量PCR和免疫印迹实验检测转染后氧化损伤的SRA01/04细胞中ERCC6 mRNA和科凯恩综合征互补B蛋白（CSB）的表达变化。采用免疫印迹实验和EdU染色实验分别检测转染后各组SRA01/04细胞凋亡相关蛋白表达变化和细胞增殖能力。结果　当H₂O₂处理浓度为200 μmol·L^-1时， SRA01/04细胞ERCC6 mRNA和CSB蛋白的相对表达量最低，后续转染实验H₂O₂处理浓度均采用200 μmol·L^-1。与H₂O₂组和H₂O₂+空白质粒转染组相比，H₂O₂+pcDNA3.1-ERCC6质粒转染组SRA01/04细胞ERCC6 mRNA和蛋白表达水平均显著增高（均为P<0.05）。进一步实验发现，与另外两组相比，H₂O₂+pcDNA3.1-ERCC6质粒转染组SRA01/04细胞中的促凋亡蛋白Bax表达均明显降低，而抑制凋亡的蛋白Bcl-2表达则均明显增加（均为P<0.05）。EdU染色实验检测结果显示,与H₂O₂+空白质粒转染组相比，H₂O₂+pcDNA3.1-ERCC6质粒转染组SRA01/04细胞内绿色荧光亮度增高，阳性细胞数量增加，表明SRA01/04细胞的增殖能力增强。结论　ERCC6基因可能通过减弱DNA的核苷酸切除修复作用抑制氧化损伤的晶状体上皮细胞增殖、促进其凋亡从而导致老年性白内障的发生。

Effect of DNA oxidative damage repair gene ERCC6 on the proliferation and apoptosis of lens epithelial cells in the H2O2-induced oxidative damage model

Objective To investigate the effect of DNA oxidative damage repair gene ERCC6 on the proliferation and apoptosis of lens epithelial cells(LECs)in the H₂O₂-induced oxidative damage model.Methods SRA01/04 cells were treated with different concentrations of H₂O₂ to establish an oxidative damage model.SRA01/04 cells with oxidative damage were divided into the H₂O₂ group,H₂O₂+blank plasmid group,and H₂O₂+pcDNA3.1-ERCC6 plasmid group for transfection.The expression of ERCC6 mRNA and Cockayne syndrome B protein(CSB)in transfected SRA01/04 cells were detected by the quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot assay.The apoptosis-associated protein expression and proliferation of SRA01/04 cells were determined by the Western blot and EdU staining.Results When the concentration of H₂O₂ was 200μmol·L^-1,the relative expression of ERCC6 mRNA and CSB protein in SRA01/04 cells was the lowest.The subsequent transfection was performed in H₂O₂ with a concentration of 200μmol·L^-1.Compared with the H₂O₂ group and the H₂O₂+blank plasmid group,the expression levels of ERCC6 mRNA and protein in SRA01/04 cells transfected with H₂O₂ and pcDNA3.1-ERCC6 plasmid were significantly increased(all P<0.05).Further experiments showed that compared with the other two groups,the expression of pro-apoptotic protein Bax in SRA01/04 cells transfected with H₂O₂ and pcDNA3.1-ERCC6 plasmid was significantly decreased,while the expression of anti-apoptotic protein Bcl2 was significantly increased(all P<0.05).The results of EdU staining showed that compared with the H₂O₂+blank plasmid group,the green fluorescence intensity and the number of positive cells in the H₂O₂+pcDNA3.1-ERCC6 plasmid group were both increased,indicating that the proliferation capacity of SRA01/04 cells was increased.Conclusion The ERCC6 gene may inhibit the proliferation of LECs by attenuating the nucleotide excision repair effect of DNA to promote their apoptosis and eventually lead to the occurrence of senile cataracts.