microRNAs在糖尿病视网膜病变模型眼内炎症反应中的调控作用

As one of the major complications of diabetes mellitus (DM), Diabetic Retinopathy (DR) has been regarded as a sort of inflammation disease, about which the mechanism is still unclear. microRNAs (miRNAs), as a post-transcriptional regulator, has rarely been investigated in ocular fundus diseases so far. A recent report showed that many miRNAs were differentially expressed in the retinas of diabetic rats and controls. Our previous publication proved that miRNA could decrease retinal neovascularization via regulating the transcription of some angiogenic growth factor genes in oxygen-induced retinopathy (OIR) mouse model. However, it is completely unknown whether miRNAs play important roles in regulating on the pathogenesis of DR particularly in inflammation response, and what the mechanism of inflammation development in DR regulated by miRNAs is. Our project is designed to use animal model and miRNAs analysis technique to roughly identify several significantly decreased miRNAs in retina from animal model, and to use modern molecular biology techniques, microsurgical techniques, histochemical techniques, and protein techniques to identify the regulating mechanism of miRNAs in the inflammation response in DR, and then to deeply explore the crucial roles of miRNAs in DR with translational medicine research. Our project will focus on exploring the close relationship between miRNAs and the pathological changes of the retinal vascular inflammation or the changes of proinflammatory factors expression in retinas from DR animal model. The present study may further clarify the regulation of miRNAs on eye inflammation in DR animal model. A novel therapeutic target at the nucleic acid level will be explored for the treatment of early DR.

糖尿病视网膜病变（DR）是糖尿病重要并发症之一。作为炎性疾病，其发病机制尚不明确。微小RNA（miRNAs）作为基因转录后调控因子，在眼底病中的作用了解甚少。有研究显示DR大鼠模型的视网膜中miRNAs表达异常。我们在氧诱导的视网膜病变小鼠模型中，发现miRNA可通过调控下游靶基因改善视网膜新生血管形成。然而，miRNAs在DR的炎症反应中是否具有调控作用，其机制如何，目前不清楚。因此，本研究拟采用模式动物和miRNAs分析技术筛选DR模型视网膜中显著下调的miRNAs，用现代分子生物学技术、显微外科、组织化学与蛋白质技术研究miRNAs在DR炎症反应中的调控作用，并对其中重要的miRNAs进行转化医学的研究。本项目将重点阐释miRNAs与DR模型视网膜血管炎症反应、促炎症因子表达之间的关系，进一步阐明miRNAs在DR炎症反应中的调控机制，并从核酸水平对DR早期防治提出科学依据。

糖尿病视网膜病变（DR）是糖尿病重要并发症之一。作为炎性疾病，其发病机制尚不明确。微小RNA（miRNAs）作为基因转录后调控因子，在眼底病中的作用了解甚少。有研究显示DR大鼠模型的视网膜中miRNAs表达异常。我们在氧诱导的视网膜病变小鼠模型中，发现miRNA可通过调控下游靶基因改善视网膜新生血管形成。然而，miRNAs在DR的炎症反应中是否具有调控作用，其机制如何，目前不清楚。因此，本研究采用链脲佐菌素(STZ)注射建立了糖尿病小鼠模型，利用real time RT-PCR技术确定在DR模型视网膜中miR-126与miR-181c显著降低；采用基因体外重组技术，构建了重组质粒载体pCMV-MIR-126和pCMV-MIR-181c；对糖尿病小鼠行质粒载体pCMV-MIR-126或pCMV-MIR-181c / 脂质体混合物玻璃体内注射，恢复了其眼内miR-126或miR-181c水平；采用显微外科、组织化学等技术分别检测糖尿病小鼠玻璃体内注射前、后视网膜微血管白细胞粘附数目、视网膜无灌注毛细血管数目，结果显示玻璃体内注射组小鼠视网膜微血管白细胞粘附数目、视网膜无灌注毛细血管数目较未注射组均显著减少；利用生物信息学分析显示，miR-181c可与促炎症因子IL-6、TNF-α的3’-UTR序列结合，miR-126可与VCAM-1序列结合；Western-blotting结果进一步证明恢复眼内miR-181c或 miR-126水平可显著降低眼内IL-6、TNF-α或VCAM-1过表达。本研究证实miRNAs对促炎症因子的表达在转录水平具有重要调控作用，保证DR模型鼠眼内的miRNAs正常水平对DR模型视网膜血管炎症反应的改善具有重要意义，也为采用核酸玻璃体内注射对DR早期防治提出了科学依据。此外，还开展了《胰岛素对糖尿病血栓形成的影响》的相关研究，结果显示：胰岛素对凝血酶诱导的血小板聚集与ATP释放具有剂量依赖的增强作用；可通过与血小板表面胰岛素受体（IR）结合，经PI3K、Akt、GSK3β、sGC等信号途径促进血小板活化；可促进凝血酶诱导的血小板表达P-选择素，具有时间依赖性；还可促进VEGF、PDGF、Endostatin在凝血酶诱导的血小板中高表达，并呈剂量依赖性。该部分研究暗示胰岛素治疗或加剧糖尿病患者血栓形成，对于DR的影响有待于下一步继续研究。