Objective To investigate the crystalline expression change induced by ultraviolet (UV) exposure in human lens epithelial cells (LECs).Methods LECs (including cells from primary culture and cell line) were exposed to UVC at the wavelength of 254 nm for certain time and were collected for the following assays:①apoptosis detection by propidium iodide (PI) staining and mitochondrial membrane potential evaluation;②quantitative PCR (qPCR) for measuring the expression of crystallin coding genes CYRAA,CRYAB and CRYBB2;③Immunofluorescence staining of CRYAA;④pyrosequencing analysis of the promoter region of CRYAA to evaluated the methylation status.Results LECs exposed to UVC at the intensity of 25 μW/cm2 for 0,10,20 and 30 min showed an increasing apoptosis rate by immediate propidium iodide (PI) staining at 0,7％,33％ and 100％.LECs exposed to UVC for 10 seconds or longer died at 100％ after 24 hours,whereas LECs exposed to UVC for 5 seconds died partially after 24 hours;the CRYAA gene expression showed a significant increase after 24 hours.The pyrosequencing of CRYAA promoter showed that the methylation status did not change in the UVC-treated cells.Conclusions The exposure to UVC for longer than 10 seconds would 100％ kill the cultured LECs.A 5 seconds-exposure to UVC would induce an increase in CRYAA expression,and the up-regulation is not the result from demethylation.%目的 研究在紫外线照射下人晶状体上皮细胞(LEC)中晶状体蛋白的表达变化.方法 用波长为254 nm的紫外线C(UVC)照射LEC(包括原代细胞和细胞系),一定时间后收集照射后的细胞进行如下实验:①碘化丙啶(PI)染色和线粒体膜电位流式检测对细胞进行凋亡评估;②实时荧光定量聚合酶链反应(PCR)检测CYRAA,CRYAB,CRYBB2基因的mRNA表达量;③免疫荧光染色检测CRYAA的蛋白表达;④焦磷酸测序检测CRYAA基因的启动子区域甲基化.结果 LEC在强度为25 μW/cm2的UVC照射0、10、20、30 min后立即做PI染色,结果显示:随着照射时间的增加,细胞凋亡率递增,分别为0、7％、33％和100％.LEC在UVC照射10 s并培养24 h后100％死亡;照射5 s并培养24 h后,部分凋亡,CRYAA基因表达量显著上调(＞4倍),但是细胞传代后CRYAA基因表达量恢复正常水平.焦磷酸测序结果显示,CRYAA基因的启动子区甲基化水平无明显变化.结论 LEC在受到强度为25 μW/cm2的UVC照射10 s并继续培养24 h后死亡,照射5s并继续培养24h后,CRYAA基因表达上调,提示其抗氧化作用,但该上调不是通过CRYAA基因启动子甲基化改变来调控的.
展开▼
