MSCs治疗干燥综合征中CD4+ T细胞miRNAs通路的作用机制

MSCs can inhibit CD4 + T cell proliferation and secretion of inflammatory cytokines, which has been clinically used to treat autoimmune diseases such as Sjogren's syndrome, but the mechanism is still unclear. Recent studies have reported that many soluble factors secreted by MSCs can change miRNA expression through different ways, thereby affecting cell function, meanwhile aberrant miRNAs in CD4 + T cells are related to autoreactive T-cell dysfunction. In this study, MSCs are in vitro co-cultured with activated peripheral CD4 + T cells from healthy volunteers and SjS patients respectively. MiRNA and target gene expression in CD4 + T cells, and possible pathways to influence from MSCs are evaluated. MiRNA overexpression or inhibition in activated CD4 + T cell is performed to determined whether MSCs suppress CD4 + T cell function via miRNAs. Furthermore, congenic bone marrow-derived MSC treatment for SjS-like mouse is carried out to investigate the miRNA, target gene expression and function of splenic CD4 + T cells and salivary gland tissues in vivo. We try to discover whether MSCs inhibit the function of CD4 + T cells via regulating miRNAs in activated CD4 + T cells, therefore, to improve the SjS autoreactive T cells.

MSCs能抑制CD4+ T细胞增殖和炎性细胞因子分泌，临床上已用来治疗干燥综合征等自身免疫病，然而其作用机制未完全清楚。近年研究报道MSCs分泌的许多可溶性因子能通过不同机制改变细胞miRNAs表达，进而影响细胞功能，而CD4+ T细胞内异常miRNAs与自体反应性T细胞功能异常相关。本研究运用MSCs分别与健康者、SjS患者活化的外周血CD4+ T细胞体外共培养，探讨其对CD4+ T细胞miRNAs和靶基因表达的影响，以及可能的影响途径，通过miRNAs过表达和抑制实验研究miRNAs对MSCs抑制CD4+ T细胞功能的影响；运用同基因骨髓MSCs治疗SjS小鼠，探讨其对体内脾脏CD4+ T细胞和唾液腺组织miRNAs和靶基因表达及功能的影响。我们试图发现MSCs是否通过调控活化CD4+ T细胞miRNAs，进而抑制CD4+ T细胞功能，达到改善SjS自体反应性T细胞异常目的。

间充质干细胞（mesenchymal stem cells，MSCs）能抑制自体免疫反应中CD4+ T细胞增殖和下游炎性细胞因子分泌，本研究通过对SS患者与NOD模型小鼠的MSCs治疗研究，试图发现MSCs是否通过调控活化CD4+ T细胞miRNAs及相关炎性因子的表达，进而抑制CD4+ T细胞功能，达到改善SS自体反应性T细胞异常目的。 . 为探讨体外MSCs对人体CD4+ T细胞的细胞因子分泌与miRNAs表达的影响，本研究首先运用hUC-MSCs分别与健康者、SS患者活化的外周血CD4+ T细胞体外共培养，发现在CD3、CD28抗体共刺激下，MSCs能抑制活化的CD4+ T细胞增殖，同时抑制CD4+ T细胞凋亡，尤其在用IFN-γ预刺激MSCs后；miRNA芯片发现MSCs能使活化的外周血CD4+ T细胞内miR-4734等表达上调，使miR-155等表达下调；MSCs共培养组和IFN-γ预刺激后MSCs共培养组上清液中PGE2等水平均有升高，提示MSCs可能通过分泌PGE2等可溶性因子改变CD4+ T细胞miRNAs表达，抑制CD4+ T细胞活化、增殖等。.对SS自发病模型NOD小鼠应用hUC-MSCs进行在体治疗实验中发现，hUC-MSCs能使小鼠脾脏CD4+ T细胞内miR-690等35个miRNAs表达下调，使miR-20a-5p等24个miRNAs表达上调；MSCs治疗组小鼠血清中IL-6、HGF、IL-10、PGE2、TGF-β1等水平均有升高，而IL-2、IFN-γ等水平下降，提示MSCs可能通过促进HGF、IL-10、PGE2、TGF-β1等抑炎因子的分泌，抑制IL-2、IFN-γ等炎症因子，调控CD4+ T细胞miRNAs表达而发挥其治疗作用。.为研究MSCs对SS患者CD4+ T细胞的免疫抑制效应, 本课题组用生物学活性更稳定的人乳牙牙髓干细胞（SHED）联合IL-6抗体或STAT3抑制剂体外作用于干燥综合征患者的PBMC中CD4+ T细胞，发现SHED可抑制SS患者PBMC中CD4+ T细胞的增殖，增加SS患者CD4+ T细胞中Treg的比例；IL-6抗体可降低与SHED共培养的SS患者PBMC中CD4+ T细胞Th2亚型比例；STAT3抑制剂可降低CD4+ T细胞Th1、Th2、Th17和Treg的比例，对Tfh比例无明显影响。