携带IGF1和Ang1基因的小鼠骨髓间充质干细胞对氧诱导ROP模型鼠的干预效果和机制研究

Retinopathy of prematurity (ROP), previously known as retrolental fibroplasia (RLF), is a vasoproliferative retinal disorder that primarily occurs in preterm infants with low birth weight. Although there has been some treatment methods such as laser photography, cryotherapy，anti VEGF therapy,ROP still constitutes to be one of the main disorders causing blindness and visual impairment in childhood. Recent researches showed that Early vascular occlusion and late vascular proliferation caused by the promoting / inhibiting angiogenesis factor imbalance may be the main pathological mechanism of ROP. IGF1 and Ang1 are important angiogenic factor, the former can promote angiogenesis through regulate the secretion and activity of VEGF, the latter is the key to the formation of normal vascular structure, while the MSCs offers much promise in the prevention and treatment of Ocular fundus disease because of its low immunity, good anti inflammatory, paracrine and implantation of the substitution effect. In this project, one new strage will developed to prevent ROP by maintain the normal vascular development strategy. .In this study, IGF1 plasmid and Ang1 plasmid will be constructed by Gateway technology respectively, then packaged with lentiviral virus and transfected into mice bone mesenchymal stem cells. The obtained stably transfected cell lines were infused to the ROP mice model by intravitreal injection. The retina structure and microvascular structure of the ROP mice model after cell transplantation were observed through morphology method, immunohistochemistry, western blotting, Q-PCR, and electron microscope scan. At the same time, the interaction between retinal stem cell and BMSCs modified by IGF1 and Ang1 gene will be investigated by co-culture experiments in vitro. Our study will be helpful to confirm the effectiveness of stem cells combined with gene therapy in ROP, and further understand the role of retinal vascular damage in mechanism of ROP, it also benifit to find more effective intervention approach for ROP.

早产儿视网膜病（ROP）是一种常见于早产、低体重儿的眼底疾病，严重者可致视网膜脱离，是儿童盲的主要原因。高氧、炎症等导致的促/抑血管生成因子调控失衡是ROP发病的核心机制。近年来以干细胞为载体的基因治疗在血管生成相关性疾病中受到高度关注。本研究打破既往以视网膜毁损或血管抑制为主的治疗策略，变堵为疏，从phase 1着手，以血管生成调控网络中对早期血管生成和血管成熟至关重要的IGF1和Ang1基因为标靶，联合骨髓间充质干细胞治疗来维持视网膜血管正常生成，通过分别构建IGF1和Ang1质粒，慢病毒介导转染MSCs后，玻璃体注射至氧诱导ROP模型鼠体内，通过形态学、免疫组化、免疫荧光、western blot、Q-PCR及电镜等观察干预后视网膜血管生成、整体结构及对相关信号通路和细胞因子表达影响，并进行体外共培养研究，旨在探讨基因联合细胞治疗ROP的可能性及确切机制，为ROP临床治疗开辟新途径。

早产儿视网膜病变（retinopathy of prematurity，ROP）是早产儿最常见的合并症之一，以视网膜血管异常增殖为特点，胎龄、体重越低，发生率越高，严重者可致视网膜脱离，是儿童致盲和视力障碍的主要原因。目前，ROP的发病机制尚未完全明确，现有治疗手段以视网膜毁损或血管抑制为主，仍难以彻底解决问题。本项目创新性提出以疏为主，通过干细胞联合基因治疗维持视网膜血管正常发育来防治ROP的新设想。通过研究，成功建立了两株携带IGF1和Ang1基因的慢病毒介导MSCs稳转细胞株，取得了较高的稳定表达效率；同时通过波动氧环境，成功建立了氧诱导ROP小鼠模型；并应用机械酶消化化法成功培养RSCs；应用BDNF进行RSC诱导可分化为视网膜神经节细胞、双极细胞，显示其可望在神经视网膜修复中发挥作用。同时，通过体外共培养和体内实验，初步证实了基因联合细胞治疗ROP的可能性，其机制可能与直接调控视网膜血管生成及诱导内源性视网膜干细胞分化有关。本课题的实施验证了我们最初提出的“化堵为疏”，以维持视网膜血管和神经正常发育，替代既往以毁损和抑制视网膜血管发育的ROP治疗手段，为未来ROP新的治疗手段提供了思路。. 在本课题资助下，已发表学术论文8篇，其中SCI论文4篇，影响因子5分以上两篇。在全国性学术会议交流论文7篇，其中2篇论文分别获得中国医师协会第八届、第九届新生儿科医师大会优秀论文奖。在投论文3篇。目前已培养毕业硕士2人，博士1人。