CAJ | 학술논문

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.
목적 탐토내질망응격중미절첩단백응답재정상체상피세포조망중적작용급기여백내장발병궤제적관계.방법 장hLECs분위대조조화실험조A、B、C、D조,분별용0、1、2、5、10mmol/L적동형반광안산(Hey)작용세포24h,응용MTT법검측불동농도약물대세포증식억제솔:응용Hoechst33258형광염색검측세포조망솔;응강활성양형광탐침DCFH-DA검측자격후세포중활성양산물(R0s);응천GSH검측시제합검측자격후세포중환원형곡광감태(GSH)적함량;응용Westernblot기술검측자격후세포중적GRP78,caspase-12적표체.결과 불동농도적Hey자격정체상피세포후,세포활력정농도의뢰성하강,기중5mmol/L,10mmol/LHey가사세포활성하강48.3%.57.7%.여대조조상비차이구유통계학의의(P<0.01),병차능유도세포적명현조망;세포중ROS수평정농도의뢰성승고,GSH감소,C、D조여대조조상비,차이구유통계학의의(P<0.05);Western bolt기술검측현시자격후세포중GRP78,emspase-12표체승고,C、D조여대조조상비,차이구유통계학의의(P<0.05).결론 고농도적내질망자격인자가자격정체상피세포산생내질망응격,병통과미절첩단백반응도치정체-망피세포조망,산생백내장.
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.