摘要
背景 年龄相关性白内障的发生、发展与长期氧化应激导致的细胞凋亡有关.研究表明,过氧化氢（H2O2）处理体外培养的细胞后可激活细胞内的黏着斑激酶（FAK）信号通路,从而影响细胞的增生与凋亡.研究FAK对氧化应激晶状体上皮细胞（LECs）的影响在年龄相关性白内障的预防方面有重要意义. 目的 研究H2O2对LECs中FAK表达的影响,探讨FAK对细胞的调控功能. 方法 用含质量分数10％胎牛血清的低糖DMEM培养液在体积分数5％CO2培养箱中常规培养人LECs株HLECs-B3,不含H2O2的为阴性对照组,在含不同浓度（30、50、70、100、300、500、700、1000 μmol/L）H2O2的培养基作用24 h,采用CCK-8法检测各组细胞的存活率；对细胞进行FITC免疫荧光染色,荧光显微镜下观察各组细胞的形态变化及细胞内FAK的表达分布；观察Hoeehst33258染色的细胞核,评估各组细胞的凋亡情况；采用Western blot法技术半定量检测各组细胞内FAK蛋白的表达量及其磷酸化水平. 结果 对照组,30、50、70、100、300、500、700、1000 μmol/L H2O2组HLECs-B3细胞的存活率分别为（1.00±0.03）％、（1.24±0.03）％、（1.36±0.24）％、（0.93±0.02）％、（1.75±0.19）％、（1.37±0.18）％、（0.64±0.01）％、（0.59±0.11）％、（0.14±0.05）％,各组间细胞存活率的差异有统计学意义（F=95.30,P=0.oo）,其中300 μmol/L以下的各H2O2组细胞存活率均明显高于对照组,而＞500 μmol/L的各H2O2组细胞存活率均明显低于对照组,差异均有统计学意义（P＜0.05）.不同浓度H2O2刺激24 h后,各组HLECs-B3细胞形态由多边形变为长梭形,并伸出伪足,FAK在细胞中呈绿色荧光,其分布随H2O2浓度的变化而发生改变.1000 μmol/L H2O2作用细胞24 h,可见细胞碎片,细胞发生凋亡.Western blot法检测发现,不同浓度H2O2处理HLECs-B3 24 h后,细胞中FAK的表达量明显不同,总体差异有统计学意义（F=28.08,P=0.00）,其中�
Background The pathogenesis of age-related cataract is associated with the apoptosis of lens epithelial cells （LECs） caused by oxidative stress.Previous studies showed that intracellular focal adhesion kinase （FAK） pathway can be activated by H2O2 in vitro,which induced apoptosis of cells.To investigate the effect of oxidative on FAK expression in LECs is one of important studies in the prevention of age-related cataract.Objective This study was to investigate the expression and function of FAK in human LECs treated by H2O2.Methods Human LECs strain （HLECs-B3） were cultured in vitro in the low glucose DMEM with 10% fetal bovine serum.Different concentrations （0,30,50,70,100,300,500,700,1000 μmol/L） of H2O2 were added into the culture medium for 24 hours.The survival rate of the cells was detected by Cell Counting Kit-8 （CCK-8） assay.Cell morphology as well as the expression and distribution of FAK in the cells were observed by immunofluorescent staining under the laser confocal microscope.Apoptosis was observed by hoechst33258 staining,and Western blot assay was used to quantitatively detect the expression and phosphorylation of FAK.Results The survival rate of the cells was （1.00±0.03） %,（1.24±0.03）%,（1.36±0.24） %,（0.93±0.02）%,（1.75±0.19）%,（1.37±0.18） %,（0.64±0.01）%,（0.59±0.11）%,（0.14±0.05）% in 0,30,50,70,100,300,500,700,1000 μmol/L H2O2 groups,with a significant difference among them （F =95.30,P =0.00）.The survival rates of the cells in the below 300 μmol/L H2O2 groups were significantly higher than those in the 0 μmol/L H2O2 group,and survival rates of the cells in the above 500 μmol/L H2O2 groups were significantly lower than those in the 0 μmol/L H2O2 group（all at P〈0.05）.After H2 O2 treatment for 24 hours,HLECs-B3 cells transformed from polygon shape to spindle shape and extended pseudopodiums,meanwhile the green fluorescence for FAK exhibited in the cytoplasm.Cell apoptosis was found in the 1000 μ mol/L H2O2 group.Western blo
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2013年第10期914-918,共5页
Chinese Journal Of Experimental Ophthalmology
基金
基金项目：广东省自然科学基金（$2011010000462）
广州市珠江科技新星专项（2011J2200050）
关键词
氧化应激
过氧化氢
晶状体上皮细胞
黏着斑激酶
Oxidative stress
Hydrogen peroxide
Lens epithelial cell
Focal adhesion kinase
