贝伐单抗对人视网膜色素上皮细胞形态、凋亡率及凋亡相关因子表达的影响  

Effects of bevacizumab on morphology and apoptosis of human retinal pigment epithelial cells as well as the expression of apoptosis-related factors

作　　者：张盼盼 董琪[2] 华英彬 徐海峰[2] ZHANG Pan-Pan;DONG Qi;HUA Ying-Bin;XU Hai-Feng

机构地区：[1]济南大学山东省医学科学院医学与生命科学学院山东省眼科研究所青岛眼科医院,山东省济南市250022 [2]山东省眼科研究所青岛眼科医院,山东省青岛市266071

出　　处：《眼科新进展》2018年第4期314-318,共5页Recent Advances in Ophthalmology

基　　金：山东省自然科学基金(编号:ZR2014HM029)~~

摘　　要：目的探讨贝伐单抗对人视网膜色素上皮细胞形态、凋亡率及凋亡相关因子表达的影响。方法用含终浓度为0.25g·L^(-1)贝伐单抗的DMEM/F12培养液培养人视网膜色素上皮细胞(ARPE-19),根据培养时间的不同分为1周加药组、2周加药组,同时设立1周对照组和2周对照组;用光镜观察各组细胞形态;采用流式细胞仪检测各组细胞凋亡情况;用实时荧光定量PCR和Western blot检测凋亡促进基因P53、TP53INP1、Bax以及凋亡抑制基因Bcl-2的mRNA和相应蛋白的表达情况。结果光镜下观察发现1周对照组和2周对照组的细胞呈典型上皮细胞形态,细胞形态呈铺路石样,单层贴壁样生长。贝伐单抗处理不同时间后,显微镜下观察发现,1周加药组和2周加药组的部分细胞形态略有改变,出现变圆的细胞,胞体拉长,边界欠清。1周对照组、1周加药组、2周对照组、2周加药组的细胞凋亡率分别为(5.57±1.46)%、(6.39±1.25)%、(6.88±1.10)%、(13.34±1.94)%,1周加药组、1周对照组和2周对照组间细胞凋亡率差异均无统计学意义(均为P>0.05);2周加药组的细胞凋亡率均高于1周对照组、2周对照组和1周加药组,差异均有统计学意义(均为P<0.05)。与1周对照组相比,1周加药组的凋亡促进因子P53、TP53INP1、Bax mRNA的表达均升高,而凋亡抑制因子Bcl-2 mRNA的表达则降低,差异均有统计学意义(均为P<0.05);2周加药组P53、TP53INP1、Bax mRNA的表达均高于2周对照组和1周加药组,而Bcl-2 mRNA的表达则降低,差异均有统计学意义(均为P<0.05);1周对照组和2周对照组相比,各因子的表达差异均无统计学意义(均为P>0.05)。各因子蛋白的表达情况与mRNA类似。结论贝伐单抗能改变ARPE49细胞的形态,上调ARPE-19细胞的凋亡率和凋亡促进因子的表达,下调凋亡抑制因子的表达,可能是抗VEGF治疗后黄斑部视网膜色素上皮层缺失的原因之一。Objective To investigate the effects of bevacizumab on cell morphology,apoptosis rate and apoptosis-related factors in human retinal pigment epitheliumcells.Methods Human retinal pigment epithelial cells(ARPE-19)were cultured in DMEM/F12 medium containing 0.25 g·L-1 bevacizumab and divided into 1-week dosing group and 2-week dosing group according to the different incubation time,respectively.Meanwhile,the control group was set up.Then,the cell morphology and apoptosis of each group were observed by light microscope and flow cytometry,accordingly.The expression levels of apoptosis-promoting genes P53,TP53INP1,Bax and apoptosis-inducible gene Bcl-2 mRNA and protein were detected by RT-PCR and Western blot,respectively.Results Light microscopic observation revealed that the cells in the 1-week control group and the 2-week control group showed typical epithelial cell morphology,and were in stone-like,single-layer adherent-like growth.Compared with the control group,part of the cell morphology after bevacizumab treatment changed slightly,and the cells became rounded,the cell body was elongated and the boundary was poor.The apoptotic rates of the 1-week control group,1-week dosing group,2-week control group and 2-week dosing group were(5.57±1.46)%,(6.39±1.25)%,(6.88±1.10)%and(13.34±1.94)%,respectively,and there was no significant difference in the apoptotic rate between 1-week control group,1-week dosing group and 2-week control group(both P>0.05),but the apoptotic rate of the 2-week dosing group was significantly higher than that of the 2-week control group,the 1-week dosing group and 1-week control group,and the differences were statistically significant(all P<0.01).RT-PCR and Western blot results showed that compared with the 1-week control group,the expression of P53,TP53INP1 and Bax mRNA was up-regulated in 1-week dosing group,but the expression of Bcl-2 mRNA was down-regulated,and the differences were statistically significant(all P<0.05).The expression levels of P53,TP53INP1 and Bax mRNA in 2-week dosing g

关 键 词：年龄相关性黄斑变性 贝伐单抗 人视网膜色素上皮细胞 细胞凋亡率 凋亡因子 

分 类 号：R774[医药卫生—眼科]