αA晶状体蛋白磷酸化对人晶状体上皮细胞抗氧化和抗凋亡能力的影响  

Roles of specific serines phosphorylation of αA crystallin on human lens epithelial cells

作　　者：叶鸿飞[1] 周鹏[1] 竺向佳[1] 蔡蕾[1] 张可可[1] 樊琪[1] 卢奕[1] 

机构地区：[1]复旦大学附属眼耳鼻喉科医院眼科,上海200031

出　　处：《中华眼科杂志》2015年第4期288-294,共7页Chinese Journal of Ophthalmology

基　　金：国家自然科学基金，上海市卫生局新百人计划，国家自然科学基金青年基金

摘　　要：目的 研究αA晶状体蛋白特定氨基酸位点磷酸化对人晶状体上皮细胞（HLEC）抗氧化及抗凋亡能力的影响.方法 实验研究.利用基因定点突变技术,将αA晶状体蛋白第45位、66位、122位丝氨酸分别突变为天冬氨酸以模拟磷酸化,导入pEGFP-C1质粒并经脂质体转染HLEC作为实验组（即S45D组、S66D组、S122D组）,转染空质粒以及过表达野生型（WT）αA晶状体蛋白的HLEC分别为空白对照组和WT组.经H2O2诱导后,分别应用超氧化物阴离子荧光探针和Annexin V-PE/7AAD双染法检测各组细胞的活性氧水平及凋亡率.两组间参数的比较采用两样本均数比较的t检验或Wilcoxon秩和检验,多组间比较采用随机区组设计的方差分析或Kruskal-Wallis检验.结果 转染模拟磷酸化αA晶状体蛋白的3组细胞超氧化物阴离子平均荧光亮度分别为167.53±18.21,143.54±4.78和143.40±30.19,较空白组（274.42±3.71）显著下降（t=9.963,P=0.001;t=34.961,P＜0.01;Z=1.993,P=0.046）,与WT组（192.67± 1.47）亦存在差异（Z=1.964,P=0.049;t=17.887,P＜0.01;Z=1.964,P=0.049）;H2O2诱导的细胞凋亡后,模拟αA晶状体蛋白磷酸化的细胞组早期凋亡率分别为15.08％±0.90％,16.59％±5.37％和15.36％±2.62％,较空白组（41.28％±2.70％;t=21.632,P＜0.01;t=8.855,P＜0.01;t=15.450,P＜0.01）及WT组（31.40％±1.64％）显著下降（t=15.117,P＜0.01;t=4.524,P=0.006;t=8.988,P=0.001）.3组实验组间相比活性氧水平及早期凋亡率均无明显差异（F=1.08,P=0.407;x2=4.345,P=0.114）.结论 αA晶状体蛋白第45位、66位、122位丝氨酸位点模拟磷酸化能增强HLEC抗氧化能力及抗凋亡活性。Objective To investigate the impacts of specific serines phosphorylation of αA Crystallin on anti-oxidation and anti-apoptotic ability of human lens epithelial cells （HLEC）.Methods Experimental Study.Using site-directed mutagenesis,serines on specific sites （Ser45,Ser66,Ser122） were replaced respectively by aspartic acid （D） to mimic phosphorylation.Target genes were inserted into pEGFP-C1 plasmid and transfected into HLEC by LipofectamineTM 2000.HLEC transfected with blank plasmid and plasmid over-expressing wide-type （WT） αA Crystallin were defined as control group and WT group.After H2O2 induction,Dihydroethidiun （DHE） and Annexin V-PE/7AAD staining were used to detect the reactive oxygen species level and apoptotic rate.T-test,Wilcoxon-two sample test,analysis of variance and Kruskal-Wallis test were used for statistical analysis.Results The DHE intensity of three phosphorylation groups were 167.53 ± 18.21,143.54±4.78 and 143.40±30.19,which reduced significantly comparing to control group （274.42± 3.71,t=9.963,P=0.001;t=34.961,P〈0.01;Z=1.993,P=0.046）;The same tendency were found when compared with cell group （WT） over-expressing wide-type αA Crystallin （192.67± 1.47,Z=1.964,P=0.049;t=17.887,P〈0.01;Z=1.964,P=0.049）.After H2O2 induction for apoptosis,early apoptotic rates for three phosphorylation groups were 15.08%±0.90%,16.59%±5.37% and 15.36%± 2.62% respectively,which significantly decreased comparing with control group（41.28%±2.70%;t=21.632,P〈0.01;t=8.855,t=15.450,P〈0.01） and WT group（31.40%± 1.64%,t=15.117,P〈0.01;t=4.524,P=0.006;t=8.988,P=0.001）.No significant differences were found in both measurements between the three phosphorylation groups（F=1.08,P=0.407;x2=4.345,P=0.114）.Conclusion The Ser45,Ser66 and Ser122 phosphorylation enhanced the anti-oxidative stress and anti-apoptotic capability of HLEC.

关 键 词：晶体 上皮细胞 活性氧 细胞凋亡 晶体蛋白质类 

分 类 号：R776.1[医药卫生—眼科]