C-Kit+/SSEA4-视网膜前体细胞与变性微环境的双向调节作用及修复视网膜的机制研究

Retinal degeneration disease (RD) is a serious blindness causing disease, and there is no effective treatment so far. Stem cell transplantation is the most promising method for the treatment of RD, and its therapeutic mechanisms include the By Stander effect, cell replacement and cell fusion. The therapeutic effect of stem cells is not only determined by its cell characteristics, but also closely related to the microenvironment of the transplated location in the recipients. Using proteomic analysis, we recently demonstrated that the exosome associated protein level was significantly increased in the degenerative retina of RCS rats, With the next generation regeneration medicine (NGRM）technology, C-Kit+/SSEA4- retinal progenitor cells (RPCs) were induced from human embronic stem cells(ESCs) which were proved to produce more lasting therapeutic effect in RD rats, it presumably related with the mutual regulation between the grafted RPCs and the degenerative microenvironment, while its mechanism has not been clarified. In present study, using secretomics, morphology and function method combined with in vitro and in vivo analysis, the interaction between RPCs and the microglial cells and Muller cells in the degenerative retinal through exosomes is investigated, the effect of gafted RPCs on the activation of the microglial cells and Muller cells in the degenerative microenvironment was especially explored. The results are expected to improve the mechanism of stem cell based cell therapy for retinal degeneration, and provide a theoretical basis for the selection of suitable stem cells and suitable time window for clinical transformation.

视网膜变性疾病（RD）是严重的致盲性眼病，目前尚无有效治疗方法。干细胞移植是治疗RD最有前景的方法，其治疗机制包括By stander效应、细胞替代和细胞融合等。干细胞的治疗作用不仅由其细胞特性所决定，还和植入处的微环境密切相关。我们采用组学分析发现，变性视网膜出现外泌体相关蛋白水平显著改变；采用NGRM技术诱导的ESCs来源的C-Kit+/SSEA4-视网膜前体细胞（RPCs）能对RD大鼠产生更持久的治疗作用，推测可能与其能和变性微环境的相互调控有关，但其机制不清楚。本研究拟采用分泌组学、形态学和功能学等方法，分析C-Kit+/SSEA4- RPCs和变性视网膜中小胶质细胞、Müller细胞通过外泌体相互作用，特别是植入的干细胞对变性微环境的主动调控作用和对细胞整合效率的影响。研究结果可望更新干细胞治疗视网膜变性的机制，为临床转化中恰当分化阶段的干细和合适时间窗的选择提供理论依据。

视网膜变性疾病（RD）是严重的致盲性眼病，目前尚无有效治疗方法。干细胞移植是治疗RD最有前景的方法，其治疗机制包括By stander效应、细胞替代和细胞融合/物质交换等。干细胞的治疗作用不仅由其细胞特性所决定，还和植入处的微环境密切相关。本课题推测，采用 NGRM 技术自 hESCs 诱导得到 C-Kit+/SSEA4- RPCs 除了更能耐受变性视网膜微环境小胶质细胞的排斥作用，还能借助于其特殊的分泌组学特征主动调节变性视网膜中的小胶质细胞和 Müller 细胞，改善变性微环境，增加整合效率，从而延长治疗效果。.为此，本研究采用分泌组学、形态学和功能学等方法，分析C-Kit+/SSEA4- RPCs和变性视网膜中小胶质细胞、Müller细胞通过外泌体相互作用的分子机制以及对视网膜变性的延缓效应。本研究首先明确了采用NGRM 技术自hESCs 诱导得到的C-Kit+/SSEA4- RPCs 的细胞生物学特性，发现这类RPCs在细胞特性上更接近原代分离的人胚胎视网膜前体细胞，C-Kit+/SSEA4- RPCs分泌的外泌体和人胚胎干细胞分泌的外泌体明显不同，内含物的主要成分为聚焦于免疫调节、代谢调控和视网膜发育的相关蛋白和 microRNA。C-Kit+/SSEA4-RPCs的外泌体可特异性调节变性视网膜中小胶质细胞的表型从激活态转变为静息态，同时可以抑制Müller细胞的胶质化，进而延缓视网膜变性过程。.通过对不同变性阶段的视网膜变性小鼠RD10视网膜的代谢组学分析发现，嘧啶代谢是RD10小鼠视网膜变性过程中变化最显著的代谢通路，其中尿苷（uridine）是最为关键的差异代谢物，尿苷的堆积是小胶质细胞激活和Müller细胞肿胀胶质化的重要微环境因素。 .C-Kit+/SSEA4- RPCs移植快速和慢速视网膜变性鼠后能通过抑制小胶质细胞激活、炎症反应和抑制Müller细胞和星形胶质细胞的胶质增生来改善视网膜变性微环境，研究发现C-Kit+RPCs发挥调节小胶质细胞免疫作用可能是通过高表达抑制小胶质细胞的相关基因，低表达小胶质募集基因实现。.本研究按计划完成了研究任务，共发表标注资助的国外SCI 论文8 篇，核心期刊1篇。参加国际会议并作大会报告1次，参加全国性会议并作大会报告6次。培养博士研究生2 名，硕士研究生2 名。