Sema3A/Nrp1通路介导Müller细胞活化参与早期糖尿病视网膜病变的作用机制

Diabetic retinopathy (DR) is one of the leading causes of preventable blindness worldwide. It has been proved that retinal neurodegeneration and microcirculatory abnormalities participate in the formation and development of early-stage DR. Müller cell activation and dysfunction play critical roles in DR, however, the underlying mechanisms in the crosstalk between neurodegeneration and microangiopathy are unclear. Previously we found that axon guidance factor Semaphorin3A (Sema3A) and its receptor neuropilin-1 (Nrp1) were up-regulated in the early-stage DR patients, and Nrp1 was highly expressed in Müller cell. Besides, Müller cell secreted several inflammatory factors and pro-angiogenic factors after treatment with recombinant Sema3A in vitro. In order to evaluate the fundamental mechanisms of Sema3A/Nrp1 pathway mediated Müller cell activation in the process of DR, the primary Müller cell and db/db diabetic mice model are used in the present study. Firstly, we aim to use lentivirus to knockdown or overexpress the Sema3A/Nrp1 gene, and evaluate the expression of immunoinflammatory factors, angiogenesis/anti-angiogenesis factors, neurotrophic factors, and oxidative stress signal pathway in Müller cell which under the stimulation of high glucose. And then, we aim to explore changes of the function in retinal ganglion cell, retinal micro-vascular endothelial cell, retina pigmented epithelial when co-cultured with gene modified Müller cell. Finally, we will perform the in vivo study to demonstrate the function of Sema3A/Nrp1 in retinal neurodegeneration and microcirculatory abnormalities in DR mice model, and explore the key regulator pathways related to Sema3A/Nrp1. This study will enrich our knowledge on the mechanisms of DR formation and explore new strategies which target Sema3A/Nrp1 signal pathway in Müller cell for early-stage DR treatment.

糖尿病视网膜病变(DR)是全球工作年龄人群重要致盲性眼病。Müller细胞活化在DR病变中有重要作用，但调控机制未明。课题组前期研究发现神经导向因子Sema3A及其受体Nrp1在DR患者组织中高表达；Nrp1与早期DR模型中的Müller细胞共定位；重组Sema3A能促进Müller细胞分泌炎症因子。为探索Sema3A/Nrp1介导Müller细胞活化在早期DR中的调控机制，本课题拟利用原代Müller细胞及db/db糖尿病小鼠，研究过表达或沉默Sema3A和Nrp1基因后，Müller细胞在高糖环境下分泌炎症因子、血管生成因子、神经营养因子的变化及潜在信号通路；视网膜血管内皮细胞、神经节细胞、色素上皮细胞与上述处理的Müller细胞共培养后的行为及功能学改变；Sema3A和Nrp1对糖尿病鼠血视网膜屏障、视网膜神经层的形态和功能影响及调控通路。该研究将为探索DR发病机制提供新的理论依据。

糖尿病视网膜病变(DR)是全球工作年龄人群重要致盲性眼病。Müller细胞活化在DR病变中有重要作用，但调控机制未明。课题拟利用原代Müller细胞及db/db糖尿病小鼠，研究过表达或沉默Sema3A和Nrp1基因后，Müller细胞在高糖环境下分泌炎症因子、血管生成因子及潜在信号通路的变化；视网膜血管内皮细胞、色素上皮细胞与高糖处理的Müller细胞共培养后的行为及功能学改变；Sema3A和Nrp1对糖尿病鼠血视网膜屏障、视网膜神经层的形态和功能影响及调控通路，为探索DR发病机制提供新的理论依据。研究发现Sema3A可以促进体外培养的Müller细胞的活化及Nrp1受体表达，促进其表达炎症因子及血管生成因子；抑制VEGF及TGF-beta诱导的血管内皮细胞及视网膜色素上皮细胞的增殖、移行、成管等细胞行为学；高糖可以诱导体外培养的Müller细胞Sema3A及Nrp1受体表达增高，可促进视网膜Müller细胞释放炎症因子；Sema3A基因过表达显著促进高糖诱导条件下Müller细胞炎症因子、VEGF165的释放和氧化应激损伤，而敲减Sema3A基因会抑制高糖诱导的Müller细胞分泌炎症因子、VEGF165，减少其氧化应激损伤。糖尿病小鼠玻璃体腔注入外源性的Sema3A可以抑制视网膜血视网膜屏障的破坏及视网膜新生血管的形成。