人脐带间充质干细胞通过保护血-视网膜屏障在治疗小鼠实验性自身免疫性葡萄膜炎中的机制研究

Uveitis is an important kind of eye disease leading to blindness, mediated mainly by CD4+ T cells. Current therapies mostly utilize hormones or immunosuppressants, with side effects inevitable. Mesenchymal stem cell (MSC) therapy is of great potential and current researches have proven the effectiveness of MSC therapy on experimental autoimmune uveitis (EAU), however, the underlying mechanism study is still at an early stage, mainly focusing on systemic T cell responses such as inhibition of T cell proliferation, decreasing the percentage of Th1，Th17 cells and increasing the percentage of Treg cells. Breakdown of the blood-retinal barrier (BRB) and infiltration of inflammatory cells into the retina are two accompanying fundamental events in the development of EAU. Only after antigen specific CD4+ T cells moved across the BRB, infiltrated the retina and were activated by local antigen presenting cells (APC), subsequent series of immune responses and tissue damage would happen. Previous studies have not yet focused on BRB and it remains largely unknown whether and how MSC can directly or indirectly protect BRB and inhibit the infiltration of immune cells. This study will utilize human umbilical MSCs to treat B10RⅢ EAU mouse model and we will firstly focus on the BRB, to evaluate the effect of MSC on BRB damage and CD4+ T cell infiltration, and to explore the regulation of MSC on cell surface adhesion molecules, cell junction molecules and chemokine receptor molecules of mouse retinal microvascular endothelial cells and CD4+ T cells. Besides, the results of mouse experiments will be further validated using human cell lines and patient-derived cells. We aim to decipher the therapy of MSC on EAU in a new perspective, and to provide further evidence to support MSC therapy on uveitis.

葡萄膜炎是一种重要致盲眼病，激素、免疫抑制剂疗法往往伴随副作用。间充质干细胞（MSC）治疗是一种潜在的新方法。已有研究表明MSC治疗小鼠实验性自身免疫性葡萄膜炎（EAU）是有效的，但机制探索还处于起步阶段，目前主要关注系统性的T细胞反应，如MSC可抑制T细胞增殖，降低Th1、Th17比例等。血-视网膜屏障（BRB）的损伤和免疫细胞的浸润在EAU的发生过程中是两个关键事件。目前尚无研究关注BRB，关于MSC能否通过直接或间接的形式保护BRB的屏障功能，抑制CD4+T细胞的浸润，以及其中的作用机制均不了解。本研究采用人脐带MSC治疗小鼠EAU，首次聚焦BRB，探索MSC对视网膜毛细血管屏障的保护作用，以及对CD4+T细胞浸润的抑制作用,探讨MSC对细胞屏障功能和T细胞浸润相关的细胞表面粘附分子、细胞连接蛋白和趋化因子受体等的影响，揭示分子机制，期望从新的角度解读MSC对EAU的治疗。

葡萄膜炎是一种主要由CD4+ T细胞介导的重要致盲性眼病，通常使用的激素、免疫抑制剂治疗往往伴随副作用。间充质干细胞（MSC）治疗是一种潜在的更安全的治疗方法。已有研究证明了MSC治疗小鼠实验性自身免疫性葡萄膜炎（EAU）的有效性，但其机制探索还处于起步阶段，且少有利用临床病人细胞进行研究的报道。血-视网膜屏障（BRB）的损伤和CD4+ T细胞的浸润在葡萄膜炎的发生过程中是关键性的事件，而关于MSC是否能够保护BRB的屏障功能，抑制CD4+ T细胞的浸润从而抑制葡萄膜炎疾病的进展尚未可知。本研究采用人脐带来源的MSC与葡萄膜炎病人外周血中分离的CD4+ T细胞进行共培养，利用RNA测序技术系统描绘了MSC共培养对CD4+T细胞转录谱的影响。通过两种重要亚型的葡萄膜炎白塞病（Behcet’s disease，BD）和Vogt-小柳原田病（Vogt-Koyanagi-Harada，VKH）的患者CD4+ T细胞进行交互验证，发现与MSC共培养后，19个基因上调，17个基因下调，其中多个差异表达基因参与免疫相关信号通路并与多种自身免疫性疾病相关。生物信息学信号通路富集分析表明，差异表达基因在TNF（Tumor Necrosis Factor，肿瘤坏死因子）信号通路中有显著富集，且这些富集的基因均呈现下降趋势，提示MSC对TNF信号通路的抑制作用。TNF信号通路是导致BRB损伤的关键因素，MSC可能通过抑制TNF信号通路，进而起到保护BRB、抑制CD4+ T细胞浸润、抑制葡萄膜炎疾病进展的作用。此外，抗TNF药物已被广泛用于顽固性葡萄膜炎患者的治疗，本研究也为利用MSC治疗葡萄膜炎提供了合理性依据。