早期糖尿病视网膜致病特异性miRNA筛查与基因调控研究

Diabetic retinopathy (DR) is one of the leading causes of blindness. There remains a critical need to better understand the underlying disease mechanisms responsible for DR in order to develop new and improved therapeutic strategies for this chronic condition. DR is a result of multiple pathogenetic processes caused by hyperglycemia and abnormalities of insulin signaling pathways , leading to retinal microvascular defects and neuroretinal dysfunction and degeneration. Although significant progress has been made, molecular mechanisms underlying the pathogenesis of DR are still not fully understood;current treatments have resulted in only a partial reduction in this risk, and the management of these conditions remains advancing need for those with diabetes. ..New insights have come from a novel family of endogenous, small (～22 nucleotides), single stranded, noncoding RNA molecules known as microRNAs (miRNAs) as developmental regulators. These molecules, only identified in humans in the last decade, modulate physiological and pathological processes by the posttranscriptional inhibition of gene expression. Many excellent performance has demonstrated miRNAs play important roles in diabetes and its complications, regulating multiple biological pathways closely related to DR . Recently, several studies have been reported about microRNAs expression in diabetic rats and found that miRNAs are involved in the pathogenesis of DR through the modulation of multiple pathogenetic pathways and may be novel therapeutic targets for the treatment of DR..However, the roles of miRNAs in DR have not been directly studied in DR patients...To further to understand the roles of miRNAs in human DR , miRNA expression profiling microarray would be used to examine the retinas of normal individuals, early diabetic without DR individuals as well as early DR individuals. By comparison of the miRNA-expression profiles, a series of miRNAs differentially expressed in the retina will be identified with Real-time PCR and in situ hybridization (ISH) for quantitative application of miRNA and cellular localization. Functional annotation analysis may reveal miRNA signatures of ongoing pathological changes in the retina of diabetic patients, which may reveal the modulating role of miRNAs in the pathogenesis of human DR. Besides, potential miRNAs will be selected as possible biomarkers, which would be used to test the peripheral blood miRNA expression of diabetic individuals without DR or with DR and target biological markers for the diagnosis of DR in its ultra-early stage might be found.

早期糖尿病视网膜病变(DR)的神经元、神经胶质细胞和微血管之间，存在复杂的网络调控机制，其分子病理扳机点至今尚未确定，转录后miRNA调控在其中发挥举足轻重的作用。课题组拟入组正常个体、早期糖尿病无DR个体以及早期DR个体，分别获取视网膜进行总RNA提取，应用人miRNA芯片杂交，分别构建miRNA表达谱，通过比对差异分析，选择差异性最显著的候选miRNA，用Real-time PCR技术进行定量，应用miRNA 原位杂交 (ISH) 技术进行细胞定位，筛选早期DR表达的视网膜特异性miRNA ，将候选miRNA 体外在细胞水平上给予干预性表达调控，进行功能分析研究，明确miRNA 的靶基因序列，探讨miRNA的基因调控机制。并将可能作为生物标记物的miRNA进行糖尿病非DR个体和DR个体外周血中miRNA的表达监测，以期获取超早期DR诊断的生物学标记物。

本研究为了在细胞水平，找到糖尿病视网膜早期损伤的生物标记物，首先搜集了老年糖尿病无视网膜病变患者8人，糖尿病早期视网膜病变老年患者8人，正常无糖尿病老年健康对照8人。细胞样品通过采集外周血单个核细胞（PBMC）和眼科手术房水离心后采集脱落细胞（细胞性质推测为视网膜神经胶质细胞或微血管上皮细胞），提取各组细胞总RNA，通过circRNA芯片杂交后，得到各自的circRNA表达谱，应用生物信息学技术进行circRNA差异表达谱的确认，以及circRNA-miRNA调控网络的预测，从而找到具有扳机点效应的circRNA和miRNA。对获取的circRNA100783进行real-time RCR定量检测，进行细胞水平的表达验证。同时，再次采集上述三组临床样本（研究组为糖尿病早期视网膜病变老年患者，阳性对照为糖尿病无视网膜病变老年患者，阴性对照为无糖尿病无视网膜病变老年患者（来自外伤的老年患者）），进行靶circRNA和靶MiRNAs的检测（包括外周血和房水），探讨这些芯片筛查的circRNA-miRNAs作为糖尿病早期视网膜病变生物标记物的可能性和可行性。已经检测了一年，随访将在近期进行。