雷帕霉素对ARPE19细胞分化的调控作用  

The pilot study on rapamycin retarding the differentiation of RPE cells in vitro

作　　者：蒋超 赵晨 石厚霞 丁思加 

机构地区：[1]南京医科大学第一附属医院眼科实验室,210029

出　　处：《中华实验眼科杂志》2015年第12期1064-1068,共5页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金青年基金项目（81222009）;江苏省科教兴卫工程重点人才基金项目（RC201149）

摘　　要：背景视网膜色素上皮（RPE）细胞移植是多种遗传性视网膜变性疾病治疗的研究方向，但体外培养的RPE细胞系都存在细胞的去分化问题，而研究表明哺乳动物类雷帕霉素靶蛋白（mTOR）信号通路的异常激活会导致RPE细胞的去分化状态。因此抑制mTOR信号通路应有助于抑制RPE细胞的去分化状态。目的观察雷帕霉素是否能够抑制mTOR信号通路，调控人RPE细胞系ARPE19细胞的分化。方法将ARPE19细胞接种于12孔板进行培养并分为对照组和雷帕霉素组，分别在培养基中添加DMSO和终浓度400nmol／L的雷帕霉素，分别于细胞培养后24h和48h收集各组细胞，通过免疫荧光法检测各组细胞中闭锁小带1（ZO-1）蛋白的表达，分别采用实时荧光定量PCR和Western blot法检测各组细胞中RPE特异性基因和蛋白的相对表达量。结果免疫荧光检测结果显示，雷帕霉素组细胞培养后24h和48h，细胞中ZO-1表达的荧光强度均明显增强。实时荧光定量PCR结果显示，雷帕霉素组ARPE19细胞处理后24h，RPE细胞特异性基因RPE65、MERKT和LRAT mRNA相对表达量较对照组分别提高了25．97％、29．71％和13．00％，差异均有统计学意义（P=0．04、0．04、0．04）；雷帕霉素组细胞处理后48h，RPE65、LRAT、rLBP1、BEST1、keratin18和MERKT mRNA的相对表达量较对照组分别提高了174．00％、88．00％、56．18％、193．81％、10．83％和35．02％，差异均有统计学意义（P=0．00、0．04、0．U1、0．04、0．04、0．03）。Western blot检测结果显示，雷帕霉素组细胞处理后24h和48h，细胞中Akt／mTOR信号通路相关蛋白p-mTOR、p-P70S6和p—S6表达条带均较对照组均明显减弱；雷帕霉素处理细胞后24h，细胞中RPE特异性蛋白ZO-1蛋白的相对表达量提高40％，差异有统计学意义（P=0．01）；雷帕霉素处理细胞后48h，ZO-1、MERKT、Catenin和LRAT蛋白的相对表达量较对照组分别提Background Retinal pigment epithelial （RPE） cell transplantation is a novel approach to the treatment of hereditary retinal diseases,however,human-derived RPE cell line occurs de-differentiation during in vitro cell culture. Studies showed that early abnormal activation of Akt/mammalian target of rapamycin （roTOR） signal pathway is the primary cause of RPE cell line de-differentiation, therefore, the inhibition of roTOR pathway will be helpful for the retard of de-differentiation of RPE cells. Objective This study aimed to investigate whether rapamycin can suppress the activation of roTOR pathway and promote differentiation of ARPE19 cells. Methods ARPE-19 cells were incubated in 12-well plate and divided into control group and rapamycin-treated group. DMSO or rapamyein with the final concentration of 400 nmol/L was added in the medium of the control group and the rapamyein-treated group, respectively. The cells of each group were collected 24 hours and 48 hours after cultured. The expression of zonula occludens-1 （ZO-1） in the cells was examined by immunofluorescence. The relative expression levels of RPE cell specific genes and proteins were assayed by using real-time quantitative PCR and Western blot,respectively. The detected results were compared between the two groups. Results ZO-1 was expressed in both group,but the fluorescence intensity was evidently enhanced in the rapamycin-treated group. The relative expression levels of RPE65, MERKT and LRAT mRNA in the cells increased by 25.97%, 29.71% and 13.00% in the rapamycin treated group compared with the control group 24 hours after cultured （P= 0.04,0.04,0. 04 ） , and the expression levels of RPE65, LRAT, rLBP1, BEST1, keratinl8 and MERKT mRNA elevated by 174.00% , 88.00% , 56. 18% ,193.81% ,10. 83% and 35.02% in the rapamycin-treated group in comparison with the control group 48 hours after cultured （ P = 0.00,0.04,0.01,0.04,0.04,0.03 ）. In addition, the expressions of p-roTOR, p-P70S6 and p-S6 protein were weaker in the rapamycin-treate

关 键 词：ARPE19细胞 雷帕霉素 哺乳动物类雷帕霉素靶蛋白信号通路 分化 

分 类 号：R774.1[医药卫生—眼科]