Smac对人晶状体上皮细胞增生的抑制作用及促凋亡作用  

The proliferative inhibition and apoptosis promotion of Smac on human lens epithelial cells

作　　者：孔德倩 郑广瑛[1] 李莉[1] 刘玥[1] 蔡芳菲[1] 

机构地区：[1]郑州大学第一附属医院眼科河南省高等学校临床医学重点学科开放实验室,郑州450052

出　　处：《中华实验眼科杂志》2017年第3期233-238,共6页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（81270986、81670836）

摘　　要：背景 晶状体后囊膜混浊（PCO）是囊外白内障摘出术后的主要并发症，其发病机制与晶状体上皮细胞（LECs）的增生、移行和上皮－间质转化（EMT）有关，探讨相关的预防和靶向治疗措施至关重要。目的 探讨Smac对人LECs增生和凋亡的作用及防治PCO的生物学作用。方法 对HLE-B3细胞株及Smac过表达人LECs株进行培养，将培养的HLE-B3细胞分为PBS组、空质粒转染组和siRNA-Smac3转染组，分别将PBS、空质粒载体和带有增强型绿色荧光蛋白（GFP）的siRNA-Smac3质粒转染细胞24 h，计算siRNA-Smac3质粒转染率。在细胞培养液中分别添加不同质量浓度（5、10、20和50 μg/ml）TGF-β2或200 μmol/L H2O2以建立PCO模型或氧化应激凋亡模型。将细胞分为PBS组、TGF-β2组和Smac过表达＋TGF-β2组，采用细胞计数试剂盒-8（CCK-8）法检测细胞增生活力；将细胞分为PBS组、H2O2组和siRNA-Smac＋H2O2组，采用流式细胞仪检测各组细胞凋亡率；分别采用实时定量PCR和Western blot法检测培养的细胞中Smac、caspase-3及增生细胞核抗原（PCNA）mRNA及蛋白的相对表达量。结果 siRNA-Smac3质粒转染细胞后GFP阳性细胞达80%以上，荧光定量PCR筛选出最佳干扰质粒为siRNA-Smac3。siRNA-Smac3转染组GFP阳性细胞率为（72.32±2.31）%，明显高于空质粒载体组的（4.91±0.24）%，差异有统计学意义（t＝116.342，P〈0.001）。LECs中Smac mRNA相对表达量为35.21±4.11，高于空质粒载体组的15.24±2.48，差异有统计学意义（t＝215.47，P〈0.05）。20 μg/ml TGF-β2作用后PBS组、TGF-β2组和Smac过表达＋TGF-β2组的细胞增生率分别为（98.4±1.7）%、（98.9±0.1）%和（64.2±3.1）%，Smac过表达＋TGF-β2组明显低于TGF-β2组，差异有统计学意义（P〈0.05）。PBS组、H2O2组和siRNA-Smac＋H2O2组细胞凋亡率分别为（2.9±1.2）%、（45.1±4.5）%和（27.5±1.8）%，siRNA-Smac＋H2O2组细胞凋亡率明显低于Background Posterior capsular opacification （PCO） is a primary complication after extracapsular cataract extraction.The mechanism of PCO is associated with proliferation, migration and epithelial-mesenchymal transition （EMT） of human lens epithelial cells （LECs）. To explore the target treatment of PCO is very important.Objective This study was to investigate the biological effects of second mitochondria-derived activator of caspases （Smac） on the proliferation and apoptosis of LECs.Methods Human LECs line （HLE-B3） and Smac-overexpressed LECs line were cultured, and the cells were transfected using small interfering RNA （siRNA）-Smac3 plasmid with green fluorescent protein （GFP） for 24 hours.Different concentration of transforming growth factor -β2 （TGF-β2） （5, 10, 20 and 50 μg/ml） or 200 μmol/L H2O2 were added respectively into the culture medium to establish PCO model and oxidative stress model.Cell counting kit-8 （CCK-8） assay was used to compare the cell proliferative activity among PBS group, TGF-β2 group and Smac-hyperexpression＋ TGF-β2 group.Flow cytometry was used to evaluate the apoptotic rate of the PBS group, H2O2 group and siRNA-Smac＋ H2O2 group.The expressions of Smac, caspase-3 and proliferating cell nuclear antigen （PCNA） mRNA and their proteins in the cells were detected by real-time quantitative PCR （RT-PCR） and Western blot.Results The GFP＋ cells were≥ 80% 12 hours after siRNA-Smac3 transfection, with the optimal plasmid of siRNA-Smac3.GFP＋ cell rate was （72.32±2.31）% in the siRNA-Smac3 transfection group, which was significantly higher than that in the blank plasmid group（[4.91±0.24]%） （t=116.342, P〈0.001）. The relevant expression levels of Smac was 35.21±4.11 in the Smac-hyperexpression group, and that in the blank plasmid group was 15.24±2.48, with a significant difference between them （t=215.47, P〈0.05）. The cell viability of 20 ng/ml TGF-β2 affected PBS group, TGF-β2 group and Smac-hyperepression＋ TGF-

关 键 词：SMAC 凋亡 增生 后发性白内障 人 晶状体上皮细胞 小干扰RNA 

分 类 号：R776.1[医药卫生—眼科]