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24 h组及造模后72 h组,每组17只,根据MiRanda、Targetscan和miRBase 3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Western blot 及实时荧光定量PCR法观察miR-181a、TNF-α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24 h和72 h显著减少,与对照组比较差异有统计学意义(P＜0.001).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24 h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,RIR 24 h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.

abstractsBackground 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 factor-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 0hour group,24-hour group and 72-hour group by random number table.Predicted target gene TNF-α was chosen,according to M iRanda,Targetscan and miRBase databases.Immunofluorescent labeling, Western blot and quantitative real-time PCR were used to identify the expression levels of miR-181a,TNF-α and RGCs.Immunofluorescent labeling of RGCs in retinal flat 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 miR181a was positively correlated with RGCs numbers (r=0.995 ,P=0.005).TNF-α and miR-181a 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 0-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-181 a.Interventions within 24 hours after reperfusion might be critical.Further study of miR181 a may help to explore new molecular targets for neuroprotection treatment.