嗅球成鞘细胞（OECs）移植恢复视网膜色素上皮细胞（RPE）功能的机制研究

Olfactory ensheathing cells (OECs) isolated from autologous olfactory bulb are specific glial subtype and are widely used in axonal regenerationin and functional recovery of injured CNS due to their huge self-renewal ability. Accordingly, the applicants transplanted OECs into retinal subspace of RCS rats, a classic animal model of retinitis pigmentosa (RP) with Mertk gene expression defects, which results in a failure of the retinal pigmented epithelium (RPE）to internalize the retinal outer segments (ROS), and found that RCS rats at 4, 8 and 12 weeks after mixture OECs/ONFs transplantation had an increased survival of photoreceptors by detecting recoverin, retinal outer segments, and PNA-Positive cones. Caspase-positive apoptotic figures in the outer nuclear layer were decreased. The upregulation of glial fibrillary acid protein （GFAP）in the Müller glia was reduced, and the b-wave of the ERG was maintained. Taken together, the process of RP was significantly slowed down and retinal degenerative events were relieved. In this project, the applicants hypothesize that OECs may improve visual function through expressing and secreting neurotrophic factor such as Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF) to protect RPE from damage, and phagocytose shed ROS, which may make RPE function and normal light transduction pathway recover. Since little is known about the mechanisms of OECs/ONFs playing important roles in retinas, the possible mechanisms of retinal pigment epithelium function recovery with the use of OECs transplantation will be studied using cell co-culture systems, Live Cell Imaging System, Electron Microscope, gene overexpression and silence technique, Immunostaining and so on. The results will provide a theoretical foundation for further clinical therapy of RP and related diseases with OECs transplantation.

嗅球成鞘细胞（OEC）是分离于自体的胶质细胞特殊亚型，以其强大的再生能力被广泛应用于中枢神经系统损伤后修复。申请者据此将其移植入MerTK基因突变所致的视网膜色素上皮细胞（RPE）吞噬功能缺陷的视网膜色素变性（RP）模型- - RCS大鼠视网膜下腔进行研究并发现：接受移植的RCS大鼠感光细胞凋亡减少，其外节长度、结构及数量明显改善，视细胞外节（ROS）堆积减少，显著延迟了RP进程。结合文献申请者推测OEC移植后视功能改善是由于其分泌神经营养因子，如脑源性神经营养因子等恢复了部分RPE功能。同时OEC发挥自身吞噬功能清除堆积的ROS碎片、减轻胶质增生反应、使光传导通路恢复正常所致。相关研究国内、外尚未见报道。本项目拟采用共培养、活细胞成像、电镜、基因过表达与沉默、免疫染色等技术系统研究并阐明OEC恢复RPE功能、吞噬ROS的分子机制，结果可为OEC移植治疗RP及其相关疾病提供直接可靠的理论依据。

研究背景：.视网膜上皮(retinal pigment epithelium,RPE) 参与视黄醇循环，吞噬脱落的光感受器细胞外节以及维持光感受器细胞兴奋性，并分泌多种生长因子。RPE一旦发生病理改变或功能紊乱，可导致多种视网膜疾病，是视功能障碍甚至是视盲的重要病因。嗅球成鞘细胞（OECs），以其强大的再生能力被广泛用于中枢神经系统损伤后修复。申请者据此将其移植入视网膜色素变性的RCS大鼠视网膜下腔进行研究并发现：接受移植的RCS大鼠感光细胞凋亡减少，其外节长度、结构及数量明显改善，视细胞外节（ROS）堆积减少，显著延迟了RP进程。.研究内容：..本项目的主要研究内容是对OECs移植改善视功能的分子机制。.重要结果、关键数据：.①成功分离、培养视网膜色素上皮细胞（RPE）和嗅球成鞘细胞（OECs），并将两者共同培养。.②研究发现OECs可能通过NGF, BDNF, bFGF对视网膜色素上皮细胞的吞噬起促进作用。.③整合素αvβ5是RPE吞噬POS的重要受体，我们成功构建了αVβ5过表达或αVβ5/siRNA载体并行慢病毒包装，感染OECs后，明显增强或降低RPE及OECs的吞噬能力。.④动物实验显示，OECs视网膜下腔移植组与PBS注射的RCS-P+大鼠相比，可见OECs在视网膜下腔及视网膜内层广泛迁移。经ERG功能检测，RCS大鼠的视功能有所恢复。.⑤利用Agilent SurePrint G3 Rat GE 8x60K Microarray进行了OECs/ONFs视网膜下腔注射修复相关眼病过程中mRNA表达差异的研究。KEGG分析显示嗅觉传导、神经活性的配体-受体相互作用、细胞黏附分子、钙信号传导通路等信号通路被明显激活。这些通路上的分子使得一些重要功能被增强，如转录因子活性、G蛋白偶联受体活性、受体活性、氧化还原酶活性等。.科学意义：.RPE功能紊乱引起的多种视网膜疾病，目前医学尚无有效疗法。我们的初步结果表明OECs移植不仅可以通过其分泌NGF, BDNF, bFGF等神经营养因子来恢复或弥补原有RPE的功能，而且可以激活多种信号传导通路，从而达到治疗或延缓一类视网膜疾病的目的，本研究通过对OECs视网膜下腔移植治疗或延缓RP进程的分子机制的研究，为临床治疗多种由RPE功能紊乱所致的疾病提供新的分子理论基础，也将会为其它各类视网膜疾病的治疗性研究带来新的启发