Rb/E2f信号通路在糖尿病视网膜神经病变发生中的作用和相关机制研究

Diabetic retinopathy (DR) is the most common complication of diabetes, and remains the leading cause of blindness among working-age individuals all over the world. Current treatments for DR (laser photocoagulation, intravitreal corticosteroids, intravitreal anti-VEGF agents and vitreoretinal surgery) are indicated in advanced stages of the disease and are associated with significant adverse effects. Therefore, new pharmacological treatments for the early stages of DR are needed. DR has been classically considered to be a microcirculatory disease of the retina. However, there is growing evidence to suggest that retinal neurodegeneration is an early event in the pathogenesis of DR, which participates in the microcirculatory abnormalities that occur in DR. Recent results also indicate that oxidative stress, DNA damage and re-entry into the cell cycle may constitute a common pathway in apoptosis in neurodegeneration diseases. Once the cell cycle is activated, the increase in the expression of transcription factor E2f1 could induce neuronal apoptosis. The features of DR, including ganglion cell death, reactive gliosis and microvascular defects, are strikingly similar with the features of Rb gene null retina. We discovered that in RbKO retinas, excitatory glutamatergic neurons (photoreceptors, bipolar and ganglion cells) undergo apoptosis, but Müller glia survive even though they also undergo ectopic division. RbKO mouse retinas also have a retinal blood vessel defect, including intraretinal hemorrhages, non-perfusion capillaries and neovascular leakages (fluorescent angiography). Thus we hypothesize that cell cycle reentry and E2f1-induced neuronal apoptosis may be the underlying mechanisms that lead to neurodegeneration of early stage of DR. We will employ streptozotocin (STZ)-induced diabetic mice, E2f1 knockout mice and inhibitors of Cdk and E2f to address how Rb/E2f pathway affects the pathogenesis of DR. Aim 1 the expression patterns of Rb/E2f pathway components, and the status of cell proliferation, cell apoptosis, ocular electrophysiological function and blood vessels in STZ-induced diabetic mouse retina. The mRNA and protein level, protein phosphorylation status of different ages will be examined by RT-PCR, Western blot and immunohistochemistry. The cdk kinase activity will be examined by radioimmunoprecipitation assay. Aim 2 the effects of E2f1 knockout on the expression patterns of Rb/E2f pathway components, and the status of cell proliferation, cell apoptosis, ocular electrophysiological function and blood vessels in STZ-induced diabetic mouse retina. Aim 3 the effects of local administration of Cdk inhibitor Cyc202 and E2f inhibitor HLM006474 on the DR phenotypes of STZ-induced diabetic mouse.

糖尿病视网膜病变是糖尿病最常见的并发症，是全球工作年龄段人群的首位致盲原因。虽然相关假说很多但其发病机制仍未完全明了，因而对其发展的控制和进行有效的治疗困难重重。糖尿病视网膜病变一直被认为是微血管病变，但近年发现其早期改变主要为视网膜神经细胞凋亡。现已知道，氧化应激导致的神经元重新进入细胞周期是神经变性疾病共同的细胞凋亡机制。我们的假说认为E2f1介导的重新进入细胞周期和细胞凋亡是早期糖尿病视网膜病变发展的重要环节。我们拟从3个方面进行研究。1.观察不同鼠龄链脲佐菌素诱导的糖尿病小鼠视网膜中细胞周期因子的表达情况、细胞增殖和凋亡、电生理功能改变、视网膜血管改变等；2.通过敲除E2f1基因观察Rb/E2f信号通路对糖尿病视网膜病变的影响；3.应用Cdk抑制剂或E2f抑制剂来观察Rb/E2f信号通路对糖尿病视网膜病变发展的影响。这项探讨糖尿病视网膜病变机制的研究结果将为此病的防治提供新策略。

糖尿病视网膜病变（Diabetic retinopathy，DR）是糖尿病最常见的并发症。本项目主要研究II型糖尿病早期视网膜神经病变的发生机制及其和Rb/E2f通路的关系。Rb基因的缺失可以导致儿童视网膜母细胞瘤，但我们发现Rb/E2f通路在DR中具有重要作用。本研究的主要研究内容和结果如下：(1) 发现啮齿动物I型糖尿病模型在4个月内并不会导致DR。通过注射STZ建立了I型糖尿病小鼠模型（C57BL）和大鼠模型（Wistar），RT-PCR检测发现它们在糖尿病早期（建模后4周前）均存在视网膜细胞周期因子表达的轻微改变，但组织学分析（建模后4-16周）、眼底荧光血管造影（建模后8-16周）均未发现视网膜神经元凋亡和微血管改变、血管渗漏等，说明在I型糖尿病建模后4个月内并不会导致DR。(2) 成功建立II型糖尿病小鼠全视网膜体外培养模型。利用C57BL小鼠和E2f1-/-小鼠建立了全视网膜体外培养模型，用（高糖+胰岛素）模拟II型糖尿病。发现II型糖尿病可导致视网膜内层氧化应激、兴奋性神经元重新进入细胞周期及凋亡（特别是双极细胞、神经节细胞）、抑制性神经元（水平细胞和无长突细胞）和Müller细胞异常增生、视网膜血管增生（自发消退被抑制）；其中兴奋性神经元重新进入细胞周期及凋亡由E2f1介导；视网膜血管自发消退被抑制可能和低氧有关。异常分裂的Müller细胞有去分化趋势。(3)RNA测序分析发现II型糖尿病明显激活了视网膜的Notch通路、AKT通路和细胞周期通路，抑制了神经突触分子的表达，这些改变和16-20周的II型糖尿病DB/DB小鼠模型视网膜非常相似。 (4) 细胞周期抑制剂（MLN4924 ）可以减少II型糖尿病导致的视网膜氧化应激、视网膜细胞异常增生和凋亡，抑制II型糖尿病导致的视网膜血管增生。..本研究发现啮齿动物I型糖尿病模型短内不会导致DR，为今后选择动物模型提供了指南。II型糖尿病主要通过氧化应激、激活E2f1通路导致神经元重新进入细胞周期及凋亡，但Müller细胞异常增生、视网膜血管增生和神经元变性并无直接关系。这些为阐明DR的发病机制提供了新的证据，同时针对细胞周期和氧化应激的药物应该是今后防治DR药物的发展方向。