未折叠蛋白应答在晶状体上皮细胞凋亡中作用的研究

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分为对照组和实验组A、B、C、D组,分别用0、1、2、5、10mmol/L的同型半胱氨酸(Hey)作用细胞24h,应用MTT法检测不同浓度药物对细胞增殖抑制率:应用Hoechst33258荧光染色检测细胞凋亡率;应刚活性氧荧光探针DCFH-DA检测刺激后细胞中活性氧产物(R0s);应川GSH检测试剂盒检测刺激后细胞中还原型谷胱甘肽(GSH)的含量;应用Westernblot技术检测刺激后细胞中的GRP78,caspase-12的表达.结果 不同浓度的Hey刺激晶体上皮细胞后,细胞活力呈浓度依赖性下降,其中5mmol/L,10mmol/LHey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;细胞中ROS水平呈浓度依赖性升高,GSH减少,C、D组与对照组相比,差异具有统计学意义(P<0.05);Western bolt技术检测显示刺激后细胞中GRP78,emspase-12表达升高,C、D组与对照组相比,差异具有统计学意义(P<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,并通过未折叠蛋白反应导致晶体-亡皮细胞凋亡,产生白内障.

The Role of Unfolded Protein Response in LECs Apoptosis

Objective To investigate the effects of the unfolded proteinresponse(UPR) in endoplasmic reticulum stress on apoptosis of human lens epithelialcells (hLECs) and the relationship between the UPR and cataract formation. Methods Cultured hLECs were divided into control group and stimulated groups-A, B, C, D, whictwere cultured in 1640 with homocysteine( 1, 2, 5, IOmmol/L) for 24h. Inhibition of cell proliferation was determined by MTT assay; cell apoptosis was detected byHoechst staining;flee glutathione was determined using a Glutathione QuantificationKit; levels of cytusolic ROS were assessed by adding H2-DCFH-DA for 20-30min followedby imaging with a fluorescent microscope;expression of Bip/GRP78, caspase-12 instimulated cells was observed by Western blot. Results After exposed to differentconcentration of Hcy, LECs showed dose-dependent decrease in cell vitality, 48.2%decline at 5mmol/L concentration of Hcy and 57.7% decline at IOmmol/L concentratioof Hey, which showed significant difference compared with control group (P <0.01),andsignificant apoptotic cells were detected;the level of ROS increased in adose-dependent manner, GSH were relatively decreased, which showed significantdifference between C,D group and control group(P<0.05); the expression of GR.P78and Caspase-12 were significant increased in C and D groups compared with controlgronp(P <0.05 ).Conclusions Higherconcentration of endoplasmic reticulumstressorscan induce endoplasmic reticulum and induce apoptosis in LECs through UPR. Theconclusion can be &awed that the UPR is probably one of initiating factors ofcataract.