Bax抑制因子1对紫外线B诱导下人晶状体上皮细胞凋亡的抑制作用  

Inhibitory effect of Bax inhibitor 1 on ultraviolet B-induced apoptosis of human lens epithelial cells

作　　者：李湘波[1] 刘艳婷 张永红 蒋殊 周克兵[1] LI Xiangbo;LIU Yanting;ZHANG Yonghong;JIANG Shu;ZHOU Kebing(Department of Ophthalmology,Nanhua Hospital Affiliated to University of South China,Hengyang 421002,Hunan Province,China)

机构地区：[1]南华大学附属南华医院眼科,湖南省衡阳市421002

出　　处：《眼科新进展》2021年第6期528-533,共6页Recent Advances in Ophthalmology

基　　金：湖南省临床医疗技术创新引导计划(编号2017SK50220)。

摘　　要：目的探讨Bax抑制因子1(BI-1)对紫外线B(UVB)诱导下人晶状体上皮细胞凋亡的抑制作用。方法取对数生长期的人晶状体上皮细胞(HLEC)系SRA01/04细胞,将BI-1过表达质粒及其空载质粒转染至SRA01/04细胞中,分别记为BI-1组、Vector组,另取不做任何处理的SRA01/04细胞作为blank组。转染48 h后,采用实时荧光定量PCR和Western blot检测转染后各组细胞中BI-1 mRNA和蛋白表达水平。而后再根据实验需要将细胞分为6组(1)Control组,SRA01/04细胞不经任何处理;(2)UVB组,采用紫外线(2.0 W·cm^(-2))处理SRA01/04细胞60 min;(3)Vector组,转染BI-1空载质粒的SRA01/04细胞;(4)BI-1组,转染BI-1过表达质粒的SRA01/04细胞;(5)Vector+UVB组,转染BI-1空载质粒的SRA01/04细胞,再经紫外线(2.0 W·cm^(-2))照射60 min;(6)BI-1+UVB组,转染BI-1过表达质粒的SRA01/04细胞,再经紫外线(2.0 W·cm^(-2))照射60 min。采用实时荧光定量PCR检测UVB照射后各组细胞中BI-1 mRNA表达水平,CCK-8法检测细胞增殖情况,流式细胞仪检测各组细胞中活性氧(ROS)含量,荧光探针法检测细胞内质网内Ca^(2+)浓度,流式细胞仪检测细胞凋亡率,Western blot检测各组细胞中相关蛋白表达水平。结果本研究中所培养的SRA01/04细胞在荧光显微镜下可观察到细胞内αA-晶状体蛋白呈红色荧光。随着UVB照射强度增加,SRA01/04细胞内BI-1 mRNA表达水平逐渐降低,本研究选择UVB照射强度为2.0 W·cm^(-2)进行后续的实验。与blank组或Vector组相比,BI-1组细胞中BI-1 mRNA和蛋白表达水平均显著上升,差异均有统计学意义(均为P<0.05)。与Control组比较,UVB组细胞增殖活性显著降低,细胞内ROS含量显著增加,细胞内质网内Ca^(2+)浓度显著升高,细胞凋亡率显著升高,细胞内Cleaved-caspase-3、Bax蛋白表达水平显著上调,Bcl-2蛋白表达水平显著下调,细胞内GRP78、p-IRE1、p-JNK和Cleaved-caspase-12蛋白表达水平显著上调(均为P<0.05)。BI-1+UVB组细胞增殖活Objective To investigate the inhibitory effect of Bax inhibitor 1(BI-1)on the apoptosis of human lens epithelial cells induced by ultraviolet B(UVB).Methods Human lens epithelial cells(HLECs)line SRA01/04 selected from logarithmic phase were transfected with BI-1 over expressed plasmid and its empty plasmid,which were respectively classified as BI-1 group and Vector group.SRA01/04 cells without any treatment were selected as the blank group.After transfection for 48 hours,the mRNA and protein expression levels of BI-1 in transfected cells of each group were detected by qRT-PCR and Western blot.The cells were then divided into six groups according to experimental needs(1)Control group,SRA01/04 cells without treatment in any way;(2)UVB group,in which SRA01/04 cells were treated with ultraviolet radiation(2.0 W·cm^(-2))for 60 minutes;(3)Vector group,in which SRA01/04 cells were transfected with BI-1 empty plasmid;(4)BI-1 group,in which SRA01/04 cells were transfected with BI-1 overexpressing plasmid;(5)Vector+UVB group,in which SRA01/04 cells were transfected with BI-1empty plasmid and irradiated with ultraviolet light(2.0 W·cm^(-2))for 60 minutes;(6)BI-1+UVB group,in which SRA01/04 cells were transfected with BI-1overexpressing plasmid and irradiated with ultraviolet light(2.0 W·cm^(-2))for 60 minutes.The expression level of BI-1 mRNA in the cells irradiated with ultraviolet light was detected by qRT-PCR,the cell proliferation was detected by CCK-8,the content of reactive oxygen species(ROS)was detected by flow cytometry,the concentration of Ca^(2+) in endoplasmic reticulum was detected by fluorescence probe method,and the apoptosis rate was detected by flow cytometry.The expression levels of related proteins in cells were detected by Western blot.Results The cultured SRA01/04 cells in this study showed red fluorescence ofαA-lens protein under fluorescence microscope.With the increase of UVB irradiation intensity,the expression level of BI-1 mRNA in SRA01/04 cells gradually decreased.UVB irradiation intensity o

关 键 词：Bax抑制因子1 紫外线B 白内障 内质网应激 

分 类 号：R776[医药卫生—眼科]