缺氧对视网膜色素上皮细胞基质细胞衍生因子-1和整合素连接激酶表达的影响  

Effect of hypoxia on expressions of stromal cell-derived factor-1 and integrin-linked kinase in retinal pigment epithelium cell in vitro

作　　者：王业青[1] 李霞[2] 吕勇[1] 王健[1] 张晓梅[1] 

机构地区：[1]哈尔滨医科大学附属第一医院眼科分院,150001 [2]哈尔滨医科大学生物信息学系,150086

出　　处：《中华实验眼科杂志》2012年第11期1013-1017,共5页Chinese Journal Of Experimental Ophthalmology

基　　金：哈医大一院科研基金资助项目（2009824）

摘　　要：背景缺氧是诱导新生血管形成的主要因素，近年来越来越多的研究证实基质细胞衍生因子-1（SDF-1）和整合素连接激酶（ILK）在新生血管性疾病中发挥着重要作用，而缺氧对视网膜色素上皮（RPE）细胞SDF-1和ILK表达的影响尚未阐明。目的探讨缺氧对体外培养RPE细胞中SDF-1和ILK表达的影响。方法从4周龄SPF级健康C57BL／6小鼠眼球中获取RPE组织并进行消化和培养，将达到80％融合的细胞进行传代。取第3代细胞制作细胞爬片后用细胞角蛋白18（CKl8）抗体进行免疫组织化学检测和鉴定，以5×104个／ml密度将RPE细胞接种于含胎牛血清的DMEM／F12培养液的培养瓶中，无血清饥饿培养24h后常氧组在常规培养基中进行培养，缺氧组在CoCl2浓度为200μmol／L的含胎牛血清的DMEM／F12培养液中进行培养，2个组根据培养时间的不同再亚分为1、3、6、12、24、48、72h组。采用逆转录PCR（RT-PCR）检测RPE细胞中SDF-1mRNA和ILKmRNA的表达，采用Westernblot检测RPE细胞中SDF-1蛋白和ILK蛋白的表达，均以目的基因A值／β-actinA值表示。结果培养的RPE细胞呈不规则多边形，细胞质内布满黑色素颗粒，90％以上的细胞对CK18呈阳性反应。随着CoCl2培养时间的延长，SDF-1mRNA和ILKmRNA的表达量逐渐增加，总体比较差异均有统计学意义（SDF-1 mRNA：F=281．875，P=0．000；ILKmRNA：F=187．566，P=0．000），12h达高峰，之后略有降低，但仍高于常氧组，缺氧1h组SDF-1mRNA及蛋白表达无明显变化；缺氧培养3、6、12、24、48、72h组SDF-1mRNA和ILKmRNA的表达量均明显高于常氧培养组，差异均有统计学意义（P〈0．01）。随着CoCl2培养时间的延长，SDF-1蛋白和ILK蛋白的表达量逐渐增加，差异均有统计学意义（SDF-1：F=44．719，P=0．000；ILK：F=144．481，P=0．000）。缺氧1h组SDF-1及ILK蛋白表达无明显变化；缺氧3、6、12、24、48、72h组SDF-1蛋�Background Hypoxia is a crucial factor of neovascularization. Many researches found that stromal cell-derived factor-1 （ SDF-1 ） and integrin-linked kinase （ILK） play an important role in the neovascular disease. However,effect of SDF-1 and ILK in eye neovascular disease is below understood. Objective The aim of this study was to investigate the effect of hypoxia on the expressions of SDF-1 and ILK in cultured retinal pigment epithelium（RPE） cells in vitro. Methods RPE tissue was isolated from 4-week-old C57BL/6 mouse and was digested and cultured in DMEM/F12 with 10% fetal bovine serum （FBS）. The cells with 80% confluence were collected and passaged. The third generation of cells were identified with cytokeratin 18 （ CK18 ） antibody by immunochemistry. The cells were inoculated at the density of 5×104 eells/ml to flee-serum DMEM/F12 for 24 hours and then were cultured in regular medium in the normoxic control group. RPE cells were cultured for 1 hour and 3,6, 12,24,48,72 hours with 200 μmol/L CoC12 in the hypoxia group. Reverse transcription-PCR（RT-PCR） was used to evaluate the expressing change of SDF-1 mRNA and ILK mRNA in RPE cells,and Western blot was used to assay the expressing change of SDF-1 protein and ILK protein in RPE ceils in different time points. The detected outcomes were represented as the ratio of target gene A value/β-actin A value. Results Cultured cells showed the polygon in shape with the black pigment granules in cytoplasm. Over 90% ceils were positive response for CK18. Expressions of the SDF-1 mRNA and ILK mRNA were increased in different time points after CoC12 co-cultured（SDF-1 mRNA：F= 281. 875, P = 0. 000 ;ILK mRNA： F = 187. 566, P = 0. 000 ） , with the highest expressing value in hypoxia at 12 hours. No significant change in the expression of SDF-1 mRNA and protein was found 1 hour after CoC12 co-cultured, but expressions of SDF-1 mRNA and ILK mRNA were significantly higher in 3,6,12,24,48 and 72 hours than the normoxic control group（ P〈0. 01 �

关 键 词：基质细胞衍生因子-1 整合素连接激酶 视网膜色素上皮细胞 CoCl2 缺氧 

分 类 号：R774.1[医药卫生—眼科]