微小RNA-181a与视网膜节细胞在视网膜缺血-再灌注中的关系及其作用机制探讨  

A pilot study on the relationship between miR-181a and RGCs in retinal ischemia-reperfusion injury model

作　　者：刘巾男[1] 何宇[2] 张军军[3] 范玮[3] 

机构地区：[1]成都市第三人民医院眼科重庆医科大学附属成都第二临床学院,成都610031 [2]四川大学华西医院西藏成办分院眼科,成都601141 [3]四川大学华西临床医学院眼科四川大学华西医院,成都610041

出　　处：《中华实验眼科杂志》2015年第11期985-990,共6页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（81070742/H1205）;四川省科技厅国际合作项目（2010HH0030）

摘　　要：背景视网膜缺血-再灌注（RIR）损伤是眼科常见病理改变之一，但其发病机制尚未明确。目的探讨大鼠RIR模型中微小RNA-181a（miR-181a）与视网膜神经节细胞（RGCs）凋亡之间的关系及可能的靶向机制。方法构建68只SD大鼠RIR模型，按随机数字表法随机分为对照组和造模后即刻组、造模后24h组及造模后72h组，每组17只，根据MiRanda、Targetscan和miRBase3个靶基因数据库的共同预测结果，选取肿瘤坏死因子-α（TNF-α）作为miR-181a的下游靶基因研究对象，通过免疫荧光标记法Westernblot及实时荧光定量PCR法观察miR-181a、TNF·仅在各组的表达情况及其与RGCs凋亡的关系。结果RGCs计数在造模后24h和72h显著减少，与对照组比较差异有统计学意义（P〈O．001）。各造模组miR-181a表达量随造模时间明显下降，与对照组比较差异有统计学意义（P〈0．05）。此外，随RIR时间的延长，miR-181a的表达量及RGCs的数量均逐渐减少，两者呈正相关（r=0．995，P=0．005）。TNF-α主要在内层视网膜表达，与miR-181a分布重叠；RIR即刻至24h之间TNF-α表达明显升高，与RGCs计数及miR-181a表达的变化趋势相反，但总体相关性分析无统计学意义。结论在RIR模型中，miR-181a可能参与RGCs凋亡的调控，TNF-α是其可能的下游靶基因之一，RIR24h前是重要的干预时机。深入研究miR-181a及其靶向基因有助于探寻新的神经保护治疗靶点。Background Retinal ischemia-reperfusion （RIR） injury is a common pathologic change. Its mechanism has not been identified. Objective This study was to investigate the relationship of microRNA-181a （miR-181a） ,tumor necrosis faetor--α （TNF-α） and retinal ganglial cells （RGCs） in RIR injury. Methods RIR models were induced in 68 rats,then the rats were randomly divided into control group and RIR groups,including 0 hour group,24-hour group and 72-hour group by random number table. Predicted target gene TNF-α was chosen, according to MiRanda,Targetscan and miRBase databases. Immunofiuorescent labeling, Western blot and quantitative real-time PC R were used to identify the expression levels of miR-181a, TNF-α and RGCs. Immunofluorescent labeling of RGCs in retinal fiat mounts was analyzed for RGCs counts. Results Compared with the control group, RGCs densitiy was obviously decreased in 24-hour and 72-hour RIR groups （P〈0. 001 ）. The expression level of mir-181a significantly decreased with reperfusion time in the RIR groups （P〈0. 05 ）. Futhermore,the expression level of miR- 181 a was positively correlated with RGCs numbers （r = 0. 995, P = 0. 005 ）. TNF-α and miR-181 a were mainly located in inner layers of retina. As opposed to the changes in RGCs numbers and miR-181a expression,TNF-α in 24-hour group was obviously higher than that of the O-hour group, though there was no statistical significance in overall correlation analysis. Conclusions In RIR,miR-181a may be involved in regulating RGCs apoptosis. TNF-α may be a target gene of miR-181a. Interventions within 24 hours after reperfusion might be critical. Further study of miR- 181 a may help to explore new molecular targets for neuroprotection treatment.

关 键 词：视网膜缺血-再灌注 视网膜神经节细胞 肿瘤坏死因子-Α 微小RNA-181a 

分 类 号：R774.1[医药卫生—眼科]