中文摘要：

背景白内障的发生与氧化应激诱导的晶状体上皮细胞（LECs）凋亡有关，而BH3-only蛋白是凋亡机制启动的关键因素，其过程可能与c—JunN末段激酶（JNK）通路激活有关，但氧化应激诱导的LECs凋亡与JNK通路的关系尚未完全阐明。目的探讨JNK／c—Jun通路及其靶基因Bim与PUMA在氧化应激诱导的LECs凋亡中的作用。方法将人LECs系HLEC—B3在含质量分数10％胎牛血清的DMEM培养基中培养和传代。将80％融合的细胞接种在24孑L板并随机分为无H2O2或H2O2（50μmol／L）处理4、8、12h组，Hoechst33258染色观察细胞形态并计数细胞凋亡率，采用Western blot法检测各组HLEC—B3细胞中JNK／c—Jun通路的激活情况和Bim、PUMA蛋白的表达水平，采用逆转录聚合酶链反应（RT—PCR）法检测c—Jun、Bim、PUMAmRNA在HLEC—B3细胞中的表达。在H2O2处理的细胞培养基中加入JNK／c—Jun通路抑制剂CEP11004或SP600125处理4h，以无H2O2或H2O2＋DMSO处理的细胞分别作为阴性和阳性对照组，Hoechst33258染色观察各组LECs的凋亡情况并检测JNK／c—Jun通路的激活情况和Bim、PUMA蛋白的表达。当培养细胞达60％融合时，将200pmol／L的Bim或PUMA小分子干扰片段转染细胞24h，然后用H2O2处理细胞采用Western blot法和RT—PCR法检测Bim、PUMA蛋白及其mRNA在HLEC—B3细胞中的表达。结果H2O2处理HLEC—B34、8、12h后，其细胞凋亡率的总体比较差异有统计学意义（F=1909．433，P=0．000），H2O2处理HLEC—B34、8、12h细胞凋亡率分别为（4．30±1．15）％、（27．08±O．74）％和（46．59±0．91）％，与无H2O2处理组（2．74±0．21）％比较差异均有统计学意义（P=0．049、0．000、0．000）。与无H2O2处理组比较，不同时间H2O2处理组JNK／c—Jun通路的激活和Bim、PUMA蛋白及其mRNA在HLEC—B3细胞中的表达强度均明显增加；加入CEP11004或SP600125后，上述因子表达下调，与DMSO培养?

英文摘要：

Background The pathogenesis and development of cataract is associated with oxidative stressinduced apoptosis of human lens epithelial cells（LECs）. BH3-only protein is a factor that can initiate apoptosis, and thus the apoptotie process is probably related to the activation of the e-Jun N-terminal kinase （JNK）. However, the relationship between oxidative stress-induced apoptosis of human LECs and the JNK pathway remains to be illuminated. Objective This study was to investigate the effects of the JNK/c-Jun pathway and its target gene,Bim （Bcl-2 interacting mediator of cell death）and PUMA （p53 up-regulated modulator of apoptosis） , on oxidative stressinduced apoptosis of human LECs. Methods The human LECs cell line（HLEC-B3） was cultured and passaged in DMEM with 10% fetal bovine serum in vitro. Confluent cells were incubated in 24 well plates and divided into 4 groups. Hydrogen peroxide（H202 ）（50 μmol/L）was used to treat the cells for 4,8 or 12 hours,and cells without H202 treatment served as the control group. Apoptosis was detected using Hoechst 33258 staining and quantified by counting the number of cells with pyknotic nuclei. In addition, confluent cells were seeded in 6 well plates, and Western blot and RT-PCR were used to detect the expression of the caspase-3, c-Jun, Bim and PUMA proteins and their mRNA in HLEC-B3 ,respectively. The JNK/c-Jun pathway inhibitors, CEP11004 or SP600125, were added into cultured media with H202 ,and ceils treated with DMSO or H202 only served as negative and positive control groups. The expression of the p-JNK, JNK, p-c-Jun, c-Jun, Bim, PUMA proteins was detected by Western blot and apoptosis was assayed using Hoechst 33258 staining. 200 pmol/L of Bim or PUMA small interference RNA（ siBim or siPUMA） fragments were transfected into the cells for 24 hours, respectively, and H2 02 was then used to treat the cells for 8 hours. The expression of the Bim and PUMA protein and their mRNA in the cells was detected by Western blot and RT-PCR,respectively.