摘要
目的探讨内质网应激（ERS）阻滞剂Salubrinal对人晶状体上皮细胞（HLEC）的保护作用及机制。方法实验研究。应用H2O2诱导HLECB，系细胞（HLE-B3）建立氧化应激模型，诱导ERS的发生。分别在H2O2干预和不干预的情况下，加入不同浓度的Salubrinal（10、15、20、25、30、35μmol／L）培养24h，采用细胞计数试剂盒（CCK-8）检测各组HLE-B，的增殖活性。实验分组为3组，A组（正常对照组）、B组（H2O2 200 μmol／L组）、C组（H2O2 200 μmol/L＋Salubrinal 25 μmol／L组）。采用原位缺口末端转移酶标记法（TUNEL）和流式细胞仪（FCM）检测药物作用48h后HLE-B，的凋亡情况。Westernblot检测不同时间点下各组葡萄糖调节蛋白质78（GRP78）、C／EBP同源蛋白（CHOP）、半胱氨酸蛋白酶12（Caspase-12）、真核细胞翻译起始因子2α（eIF2α）的磷酸化型p-elF2α的变化。单因素方差分析用于组间均数的多重比较，当方差齐时组间两两差异比较采用Dunnettt检验，当方差不齐时Dunnett’s T3用作两两比较。结果CCK-8试剂盒结果显示，没有H2O2干预时，不同浓度的Salubrinal对HLE-B，的细胞活性无明显抑制作用，存活率分别为（98．6±3．3）％，（98．7±2．6）％，（9914±3．2）％，（98．6±1．9）％，（98．8±2．5）％，（99．3±3.2）％，（99．5±2．4）％，差异无统计学意义（F=0．09，P=0．10）；应用H2O2干预时，随着Salubrinal浓度的升高，HLE-B，存活分别为（52．9±4．7）％，（65．0±3．6）％，（72．9±3．8）％．（84．5±3．6）％，（91．6±2．1）％，（93．1±2．9）％，（92．0±3.3）％，与对照组相比，差异有统计学意义（P值均〈0．01），当Salubrinal浓度大于25μmol／L时，HLE-B，细胞的存活率增加不明显（P=0．56，0．88）。流式细胞仪检测3组的细胞凋亡率分别为（1．9±0．7）％、（8．8±0．5）％、（4．3±0.3）％，各
Objective To study the protective effect of Salubrinal on human lens epithelial cells and its mechanism in endoplasmic reticulum stress （ERS）. Methods Hydrogen peroxide （H202 200 μmol/L） was used to intervene in the cultured human lens epithelial cells B3 （HLE-B3） so as to create an oxidative stress model and induce ERS in the model. Different concentration of Salubrinal （10, 15, 20, 25, 30 and 35 μmol/L） were added to the cultured HLE-B3 with or without H2O2 intervention. Then the cells were cultured for 24 hours. The cell counting kit （CCK-8） assay was used to test the viability of HLE-B3. The HLE-B3 cells were divided into three groups： Group A （normal control group）, Group B （H202 200 μmol/L group）, and Group C （H202 200 μmol/L ＋ Salubrinal 25 μmol/L group）. After 48 h, TUNEL and flow cytometry assay （FCM） were used to examine the effect of Salubrinal on HLE-B3 apoptosis. The expression of glucose- regulated protein 78（GRP78）, C/EBP homologous protein （CHOP）, cysteinyl aspartate specific proteinase 12 （Caspase-12） and phosphorylation eukaryotic translation initiation factor 2α （p-elF2α） were tested by western blot at different points in time. Data from different groups was analyzed by one-way analysis of variance （ANOVA） while Dunnett t test was used under an equal condition, Dunnett＇s T3 for the unequal variances. Results CCK-8 results showed that without the intervention of H202, different concentrations of Salubrinal had no inhibitive effect on HLE-B3 viability, and that survival rates were （98.6±3.3）%, （98.7± 2.6）%, （99.4± 3.2）%, （98.6± 1.9）%, （98.8±2.5）%, （99.3±3.2）% and （99.5±2.4）%. There was no statistically significant difference between them （F=0.09, P=0.10）. With the increasing of Salubrinal concentration, the survival rates of HLE-B3 in the presence of H2O2 intervention were （52.9±4.7）%, （65.0± 3.6）%, （72.9±3.8）%, （84.5±3.6）%, （91.6±2.1）%, （93.1±2.9）%, （92.0±3
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2016年第6期437-443,共7页
Chinese Journal of Ophthalmology
基金
国家自然科学基金（81270986）
关键词
晶体
上皮细胞
内质网应激
细胞凋亡
桂皮酸盐类
Lens, crystalline
Epithelial cells
Endoplasmic reticulum stress
Apoptosis
Cinnamates
