GJA3基因突变介导细胞凋亡引起遗传性白内障机制研究

Apoptosis analysis and functional study for hereditary congenital cataract due to GJA3(p.P187S)

目的:前期研究证实,缝隙连接蛋白基因46(GJA3)的第187位氨基酸由脯氨酸突变为丝氨酸即GJA3(p.P187S)是一遗传性白内障致病基因,本研究旨在对该基因突变进行功能分析及凋亡机制研究。方法:构建野生型GJA3和突变型GJA3(p.P187S)真核表达载体,分别转染人宫颈癌细胞(Hela)细胞进行表达,比较两组缝隙连接盘的表达差异;运用Western-Blot方法分析两组蛋白表达水平上的差异;通过TUNEL染色及流式细胞技术检测两组凋亡率差异。结果:GJA3和突变型GJA3(p.P187S)组缝隙连接盘形态观察发现无明显差异。两组蛋白的表达水平无明显差异。凋亡检测结果显示GJA3(p.P187S)突变体组凋亡率较高。结论:GJA3(p.P187S)突变未影响GJA3蛋白的表达水平、蛋白亚细胞定位及缝隙连接盘形成,但可引起细胞凋亡率上升。这提示异常凋亡可能为GJA3(p.P187S)突变导致遗传性白内障的致病机制,验证这一点需进一步研究工作的开展。

Objective:This study aimed to identify the apoptosis analysis and functional study associated with autosomal dominant congenital cataracts due to GJA3(p.P187S).Methods:The wild type and GJA3(p.P187S)mutant type were transfected into Hela cells to determine the subcellular localization and by applying Western blot analysis to determine the expression.Flow cytometry and TUNEL was used to compare the apoptosis differences between the wild type and GJA3(p.P187S)mutant GJA3.Results:It was found that the GJA3(p.P187S)mutant was aberrantly located in the cytoplasm instead of in the plasma membrane.Western blot analysis revealed that WT-GJA3 and GJA3(p.P187S)had similar protein levels in western blot analysis of the cell lysates,indicating that mutation did not result in the expression or instability of the protein.Flow cytometry and TUNEL staining showed apoptosis rate in GJA3(p.P187S)mutant group is higher than WT-GJA3group.Conclusion:The results above suggests that the apoptosis may be the pathogenic mechanism of the mutant GJA3(p.P187S)of inherited cataract,further experiments will be conducted to verify this conclusion.