IFN-γ和TNF-α诱导的骨髓间充质干细胞的免疫抑制作用及环孢素A对其的影响  

The effect of CsA on the immunosuppression of bone marrow-derived mesenchymal stem cells induced by IFN-γ and TNF-α

作　　者：唐礼功[1] 井莹莹[2] 谢森[1] 陈琪[1] 李志雄[1] 韩志鹏[2] 蒋国成[2] 刘燕[2] 

机构地区：[1]广州军区武汉总医院泌外科,武汉430070 [2]第二军医大学附属东方肝胆外科医院肿瘤免疫实验室

出　　处：《中华器官移植杂志》2010年第4期238-241,共4页Chinese Journal of Organ Transplantation

摘　　要：目的 观察γ干扰素（IFN-γ）和肿瘤坏死因子α（TNF-α）诱导的骨髓问充质干细胞（MSC）的免疫抑制作用及环孢素A（CsA）对其的影响.方法 取Balb/c小鼠骨髓,分离、培养、纯化及扩增MSC,分别以IFN-γ、TNF-α和IFN-γ＋TNF-α（终浓度均为20 ng/ml）预处理MSC 12 h,然后与Balb/c小鼠脾细胞共培养,脾细胞以刀豆蛋白A激活72 h.混合细胞培养实验分8组进行：（1）脾细胞组;（2）MSC组,MSC＋脾细胞;（3）IFN-γ处理组,IFN-γ预处理的MSC＋脾细胞;（4）TNF-α处理组,TNF-α预处理的MSC＋脾细胞;（5）联合处理组,IFN-γ与TNF-α联合预处理的MSC＋脾细胞;（6）CsA组,CsA＋脾细胞;（7）CsA联合预处理组：IFN-γ与TNF-α联合预处理的MSC＋CsA＋脾细胞;（8）1400W组,IFN-γ与TNF-α联合预处理的MSC＋脾细胞＋1400W[诱导型-氧化氮合酶（iNOS）抑制剂].各组脾细胞数量均为5000个,MSC与脾细胞按1：10混合,CsA浓度为10μg/ml.培养72 h后,以四甲基偶氮唑盐法检测脾细胞增殖情况,同时以碘化丙啶和Annexin-V凋亡双染试剂盒检测脾细胞的凋亡情况;以实时聚合酶链反应法检测经炎症因子处理的MSC的iNOS mRNA表达情况,观察CsA对MSC的iNOS mRNA表达的影响.结果 MSC组、IFN-γ处理组和TNF-α处理组的MSC对脾细胞的增殖无明显抑制作用（P〉0.05）,而联合处理组的脾细胞增殖明显受到抑制,CsA组的脾细胞增殖同样受到抑制,但IFN-γ与TNF-α联合预处理MSC产生的这种免疫抑制作用却可以被CsA所抑制（CsA联合预处理组,P〈0.05）.联合处理组、CsA组和CsA联合预处理组的脾细胞凋亡率明显高于脾细胞组、MSC组、IFN-γ处理组和TNF-α处理组（P〈0.05）,而联合处理组和CsA组的脾细胞凋亡率又明显高于csA联合预处理组（P〈0.05）.野生型MSC、分别以IFN-γ或TNF-α单独刺激的MSC均不表达iNOS mRNA,而以IFN-γ和TNF-a联合处理的MSC则高表达iNOS mRNA,但这种高表达可被CsA所�Objective To investigate the effects of cyclosporine A on the immunosuppression of bone marrow-derived mesenchymal stem cells （MSCs） induced by interferon-γ （IFN-γ） and tumor necrosis factor-α （TNF-α）.Methods MSCs were isolated, cultured, purified and proliferated from bone-marrow of Balb/c mice.After pretreated with the inflammatory cytokines IFN-γ, TNF-α and IFN-γ＋ TNF-α （final concentration of 20 ng/ml）, MSCs were co-cultured with spleen cells which were activated by concanavalin A for 72 h.Following groups were assigned： group 1, spleen cells alone; group 2, MSCs ＋ spleen cells; group 3, MSCs （pretreated with IFN-γ） ＋ spleen cells; group 4, MSCs （pretreated with TNF-α） ＋ spleen cells; group 5, MSCs （pretreated with IFN-γ and TNF-α） ＋ spleen ceils; group 6, CsA ＋ spleen cells; group 7, MSCs （pretreated with IFN-γ and TNF-α） ＋ CsA ＋ spleen cells; group 8, MSCs （pretreated with IFN-γ and TNF-α） ＋ 1400 W＋ spleen cells.The ratio of MSCs and spleen cells was 1 ： 10 with the amount of spleen cells being 5000 and the concentration of CsA 10μg/ml.The proliferation of spleen cells was accessed with MTT assay and the apoptosis was detected by flow cytometry after 72 h.Real-time PCR was used to detect the expression of iNOS mRNA in MSCs treated by inflammatory eytokines to observe influence of CsA on the expression of iNOS mRNA.Results In MSCs pretreated with or without IFN-γ, TNF had no significant inhibitory effects on the proliferation of spleen cells （P〉0.05）.However, IFN-γ in combination with TNF-α, and CsA alone could both inhibit the proliferation of spleen cells, but inhibitory effect of the former could be attenuated by the latter one （P〈0.05）.The apoptosis rate in groups 5, 6, and 7 was higher than in groups 1, 2, 3, and 4 （P〈0.05）, and that in group 5, and 6 significantly higher than in group 7 （P〈0.05）.MSCs pretreated with combination of IFN-γ and TNF-α expressed high level of iNOS than in control groups,

关 键 词：间质干细胞 干扰素Ⅱ型 肿瘤坏死因子α 免疫抑制法 环孢菌素 

分 类 号：R285.5[医药卫生—中药学]