未折叠蛋白应答在晶状体上皮细胞凋亡中的作用  

Role of the unfolded protein response in apoptosis of lens epithelial cells

作　　者：杨雪莉[1] 蔡可丽[1] 高雪[2] 吴晓莉[3] 卓建[1] 杨博[1] 刘德荣[1] 王荣[4] 

机构地区：[1]山东大学齐鲁医院眼科,济南250012 [2]山东大学第二医院眼科,济南250033 [3]山东省荣军总医院眼科,济南250013 [4]山东大学齐鲁医院教育部和卫生部心血管重构与功能研究重点实验室,济南250012

出　　处：《山东大学学报（医学版）》2008年第1期52-56,共5页Journal of Shandong University:Health Sciences

基　　金：山东省科技攻关计划(项目编号:2007GG30002009)

摘　　要：目的探讨内质网应激中未折叠蛋白应答在晶状体上皮细胞(hLECs)凋亡中的作用及其与白内障发病机制的关系。方法将hLECs分为对照组和实验A、B、C、D组,分别用0、1、2、5、10 mmol/L的同型半胱氨酸(Hcy)作用细胞24 h,应用MTT法检测不同浓度药物对细胞的增殖抑制率;应用Hoechst33258荧光染色检测细胞凋亡率;应用活性氧荧光探针DCFH-DA检测刺激后细胞中活性氧产物(ROS);应用GSH试剂盒检测刺激后细胞中还原型谷胱甘肽(GSH)的含量;应用Western blot技术检测刺激后细胞中的GRP78和Caspase-12的表达。结果不同浓度的Hcy刺激晶体上皮细胞后,细胞活力呈浓度依赖性下降,其中5 mmol/L,10 mmol/L Hcy可使细胞活性下降48.3%,57.7%,与对照组相比,差异具有统计学意义(P<0.01),并且能诱导细胞的明显凋亡;与对照组相比,C、D组细胞中ROS水平呈浓度依赖性升高,GSH含量减少,GRP78和Caspase-12表达显著升高(P均<0.05)。结论高浓度的内质网刺激因子可刺激晶体上皮细胞产生内质网应激,并通过未折叠蛋白应答导致晶状体上皮细胞凋亡,产生白内障。Objective To investigate the effects of the unfolded protein response（UPR） in endoplasmic reticulum stress on apoptosis of human lens epithelial cells （hLECs） and the relationship between UPR and cataract formation. Methods Cultured hLECs were divided into the control group and stimulated groups A, B, C and D, which were cultured in 1640 with homocysteine （ 1, 2, 5 and 10 mmol/L） for 24 h. Inhibition of the cell proliferation was determined by MTF assay; cell apoptosis was determined by Hoechst staining; free glutathione was determined using a Glutathione Quantigieation Kit; the levels of eytosolie ROS were determined by adding H2 -DCFH-DA for 20 to 30min followed by imaging with a fluorescent microscope; and the expressions of Bip/GRP78 and Caspase-12 in stimulated cells were determined by Western blot. Results After being exposed to different concentrations of Hcy, LECs showed a dose-dependent decrease in cell vitality： 48.2% dechned at 5 mmol/L concentration of Hcy and 57.7% dechned at 10 mmol/L concentration of Hcy, which showed significant differences compared with the control group （ P 〈 0.01 ）. Also significant apoptotic ceils were determined. Compared with the control group, the level of ROS increased in a dose-dependent manner and of GSH was decreased in the C and D groups（ P 〈 0.05）, also the expressions of GRP78 and Caspase-12 were significantly increased in the C and D groups （ P 〈 0.05）. Conclusions High concentration of endoplasmic reticulum stressors can induce the endoplasmic reticulum and induce apoptosis of LECs through UPR. The conclusion can be drawn that UPR is probably one of the initiating factors of cataracts.

关 键 词：白内障 发病机制 内质网应激 未折叠蛋白应答 细胞凋亡 

分 类 号：R776.1[医药卫生—眼科]