Trim9调控视网膜Müller细胞向神经节细胞定向分化  

Trim9 regulates the directional differentiation of retinal Müller cells to retinal ganglion cells

作　　者：李金香 曾琦[1] LI Jinxiang;ZENG Qi(Department of Ophthalmology,First Hospital Affiliated with Hunan Normal University(Hunan Provincial People’s Hospital),Changsha 410005,China)

机构地区：[1]湖南师范大学第一附属医院(湖南省人民医院)眼科,长沙410005

出　　处：《中南大学学报（医学版）》2023年第10期1561-1571,共11页Journal of Central South University ：Medical Science

基　　金：湖南省卫生健康委员会科研计划项目(20200757);湖南省自然科学基金(2021JJ30397);湖南省科技厅重点研发项目(2020SK2119)。

摘　　要：目的:青光眼是不可逆性致盲性眼病的主要病因,目前尚无有效疗法逆转青光眼的视觉系统损害。最近发现干细胞疗法有望使受损的视网膜神经元修复和再生,但是在干细胞来源方面仍然存在较大挑战。本研究探寻一种将视网膜Müller细胞去分化为视网膜干细胞,进一步高效定向分化为视网膜神经节细胞的方案,以期为青光眼的干细胞治疗提供新的细胞获取途径。方法:用表皮细胞生长因子和成纤维细胞生长因子2诱导大鼠视网膜Müller细胞去分化为视网膜干细胞。构建Trim9过表达慢病毒(PGC-FU-Trim9-GFP),感染由Müller细胞诱导去分化而来的视网膜干细胞,通过荧光显微镜观察和流式细胞术评估病毒感染效率。用视黄酸和脑源性神经营养因子处理过表达或未过表达Trim9的视网膜干细胞以诱导其分化为神经元和神经胶质细胞。采用免疫荧光、PCR/real-time RT-PCR和蛋白质印迹法检测各细胞标志物(GLAST、GS、rhodopsin、PKC、HPC-1、Calbindin、Thy1.1、Brn-3b、Nestin、Pax6)的表达。结果:表皮细胞生长因子和成纤维细胞生长因子2处理后的大鼠视网膜Müller细胞表达视网膜干细胞标志物Nestin和Pax6。视黄酸和脑源性神经营养因子处理后的过表达Trim9的视网膜干细胞Thy1.1阳性细胞明显增多,表明其定向分化为视网膜神经节细胞。结论:本研究成功将大鼠视网膜Müller细胞去分化为视网膜干细胞,并发现Trim9可有效促进由视网膜Müller细胞去分化而来的视网膜干细胞定向分化为视网膜神经节细胞。Objective:Glaucoma is a leading cause of irreversible blindness,and effective therapies to reverse the visual system damage caused by glaucoma are still lacking.Recently,the stem cell therapy enable the repair and regeneration of the damaged retinal neurons,but challenges regarding the source of stem cells remain.This study aims to investigate a protocol that allows the dedifferentiation of Müller cells into retinal stem cells,following by directed differentiation into retinal ganglion cells with high efficiency,and to provide a new method of cellular acquisition for retinal stem cells.Methods:Epidermal cell growth factor and fibroblast growth factor 2 were used to induce the dedifferentiation of rat retinal Müller cells into retinal neural stem cells.Retinal stem cells derived from Müller cells were infected with a Trim9 overexpression lentiviral vector(PGC-FU-Trim9-GFP),and the efficiency of viral infection was assessed by fluorescence microscopy and flow cytometry.Retinoic acid and brain-derived neurotrophic factor treatments were used to induce the differentiation of the retinal stem cells into neurons and glial cells with or without the overexpression of Trim9.The expressions of each cellular marker(GLAST,GS,rhodopsin,PKC,HPC-1,Calbindin,Thy1.1,Brn-3b,Nestin,Pax6)were detected by immunofluorescence,PCR/real-time RT-PCR or Western blotting.Results:Rat retinal Müller cells expressed neural stem cells markers(Nestin and Pax6)with the treatment of epidermal cell growth factor and fibroblast growth factor 2.The Thy1.1 positive cell rate of retinal stem cells overexpressing Trim9 was significantly increased,indicating their directional differentiation into retinal ganglion cells after treatment with retinoic acid and brain-derived neurotrophic factor.Conclusion:In this study,rat retinal Müller cells are dedifferentiated into retinal stem cells successfully,and Trim9 promotes the directional differentiation from retinal stem cells to retinal ganglion cells effectively.

关 键 词：青光眼 MÜLLER细胞 Trim9 视网膜干细胞 视网膜神经节细胞 

分 类 号：R775[医药卫生—眼科]