胰岛间充质干细胞通过外泌体内源性反转录病毒抗原诱导NOD鼠自身免疫反应

Islet mesenchymal stem cells induce autoimmune response in NOD mice by secreting exosomes that express endogenous retrovirus antigens

目的:探讨Ⅰ型糖尿病(T1D)NOD小鼠胰岛组织中诱导自身免疫反应的初始抗原及其特异性免疫反应。方法:分离NOD小鼠胰岛细胞进行体外培养,检测胰岛细胞表达间充质干细胞(MSCs)标记分子的情况,同时检测胰岛组织中MSCs和淋巴细胞的分布。收集胰岛MSCs的培养上清,并分离外泌体,将外泌体与NOD小鼠脾细胞混合共培养,检测IFN-γ的释放及B细胞增殖情况。Western blot及RT-PCR检测内源性反转录病毒(ERV)的核抗原(Gag)在胰岛MSC或其外泌体中的表达。结果:体外培养小鼠胰岛组织可生产大量MSCs,这种原代培养的细胞表达Sca-1和CD105分子。培养上清含外泌体,且该外泌体能刺激部分B细胞增殖并激活T细胞分泌IFN-γ。抗原成分分析表明NOD鼠而非B6鼠的胰岛MSC及其外泌体表达ERV Gag抗原,且该抗原基因的表达仅局限于胰岛的CD45-Sca-1+MSC,而非浸润胰岛的CD45+淋巴细胞。结论:NOD鼠胰岛干细胞分泌的外泌体表达ERV Gag抗原,并具有强的免疫原性,提示外泌体可能模拟类病毒诱导胰岛局部的免疫刺激应答,为进一步研究ERV在诱发T1D发病中的机制奠定基础。

Objective:To study the primary antigens that activate the islet-specific autoimmune response in type 1 diabetes(T1D)NOD mouse islet tissues and the antigen specificity.Methods:NOD mouse islet cells were isolated and expanded in vitro.Expressions of mesenchymal stem cell(MSCs)markers in islet cells were analyzed,and the distribution of islet MSCs and lympho⁃cytes was also detected.Culture supernatant of islet MSCs was collected for isolating exosomes,and the exosomes were co-cultured with NOD mouse spleen cells,followed by measuring release of IFN-γand proliferation of B cells.Western blot and RT-PCR were per⁃formed to detect expression of endogenous retrovirus(ERV)nuclear antigen(Gag)in islet MSCs or exosomes.Results:A large num⁃ber of MSCs could be produced from mouse islet tissue culture in vitro,and these primary cultured cells express scA-1 and CD105 mol⁃ecules.The culture supernatant contains exosomes,and the exosomes could stimulate B cells to proliferate and activate T cells to se⁃crete IFN-γ.Analysis of antigen composition showed that NOD-derived islet MSCs and exosomes could express ERV Gag antigen,which is absence in the B6-derived MSCs and exosomes.Furthermore,expression of Gag antigen is limited to the CD45-Sca-1+MSCs in the islets,but not the CD45+islet-infiltrating lymphocytes.Conclusion:Exosomes secreted by islet stem cells in NOD mice can ex⁃press ERV Gag antigen and show strong immunogenicity,suggesting that the exosomes may act as pseudo viruses to trigger islet-specific autoimmune response and diabetes in NOD mice,which lays a foundation for further research of the mechanism of ERV inducing T1D.