γ干扰素体外诱导骨髓间充质干细胞的IDO表达及其免疫调节作用

γ-interferon-induced IDO expression of mesenchymal stem cells in vitro and the immuno-regulatory mechanism

目的探讨γ干扰素（IFN-γ）体外诱导作用成人骨髓间充质干细胞（MSCs）的吲哚胺2，3-过氧化酶（IDO）的表达及IDO对异基因T淋巴细胞增殖的影响。方法从人骨髓中分离、培养MSCs，观察传代3次以上的细胞形态，应用流式细胞术检测其表面标志，以鉴定其纯度。分别用浓度为0、20、50、100、200U／ml的IFN-γ诱导作用MSCs后，以逆转录聚合酶链反应检测IDOmRNA的表达，蛋白印迹法检测IDO蛋白的表达，反相高效液相色谱法检测色氨酸的特异代谢产物犬尿氨酸的特异代谢率，以此判断IDO的活性。以丝裂霉素C灭活的外周血单个核细胞为刺激细胞，异基因T淋巴细胞为反应细胞，与经200U／ml IFN-γ作用的MSCs组成混合淋巴细胞培养体系，MSCs与反应细胞的比例分别为1：5、1：10、1：50及1：100，部分混合淋巴细胞培养体系中加入IDO的特异性抑制剂1-甲基色氨酸（1-MT），检测T淋巴细胞增殖率及培养体系中IDO活性。结果MSCs培养传代3代以后的纯度达到95％。在无IFN-γ作用时，MSCs没有IDO mRNA表达，经不同浓度的IFN-γ诱导后，均可检测到IDOmRNA表达，且表达强度呈IFN-γ剂量依赖性；IDO蛋白的表达与IDOmRNA一致；随着IFN-γ浓度的增加，MSCs培养上清液中的色氨酸浓度降低，而犬尿氨酸浓度升高。在无MSCs的混合淋巴细胞培养体系中，T淋巴细胞的增殖率为（80．2±5．7）％，当MSCs与反应细胞之比为1：5、1：10、1：50及1：100时，T淋巴细胞的增殖率分别为（28．1±3．2）％、（38．6±4．0）％、（57．2±2．5）％和（84．1±4．6）％，若在相同的混合淋巴细胞培养体系中加入1-MT，则MSCs对T淋巴细胞增殖的抑制作用基本消失；培养体系中IDO的活性与MSCs的数量成正比。结论γ干扰素在体外能诱导MSCs的IDO表达，其表达水平与γ干扰素呈剂量依赖性；IDO能抑制T淋巴细胞增殖。

Objective To study the effect of y-interferon （IFN-γ） on the IDO expression of mesenchymal stem cells （MSCs） and the effect of IDO activity in MSCs on allogeneic T-lymphocyte proliferation, and explore the immuno-regulatory mechanism of MSCs. Methods MSCs were isolated and cultured from human bone marrow cells. The morphology of more than 3 passage MSCs was observed by microphotograph, and the purity of MSCs was identified with phenotypes tested by flow cytometry. The expression of IDO mRNA and protein and IDO functional activity in MSCs induced by IFN-γ at various concentrations （0, 20, 50, 100, 200 U/ml） were detected by RT-PCR method, Western blot method and HPLC method respectively. MLRs cultures were set up with mitomycin C-treated human peripheral blood mononuclear cells （PBMCs） as stimulators and human allogeneic T- lymphocytes as responder cells. In MSCs/MLRs coculture experiments, MLRs were performed on a layer of 1×10^5s, 5×10^4, 1×10^4 or 5×10^3 MSCs （MSCs：responderTcells =1：5, 1：10, 1：50, 1： 100） in the presence or absence of 1-methyl-D-trytophan （1-MT）. T-lymphocytes proliferation and IDO activity in coculture supernatant were determined. Results The expression of IDO mRNA and protein expression and IDO functional activity were found in MSCs induced by IFN-γ, and the amount of expression and ff）O activity were increased gradually with the increases of concentrations of IFN-γ; When cocultured with 1 × 10^5 , 5 × 10^4, 1 × 10^4 MSCs （MSCs： responder T cells = 1：5, 1 ： 10, 1：50）, T-lymphocytes proliferation was decreased apparently and ff）O activity increased obviously as compared with control group （P〈0. 05）. In parallel experiments, 1-MT was added. T-lymphocytes proliferation and ff）O activity restored partially. While cocultured with 5 × 10^3 MSCs （MSCs： responder T cells = 1 ： 100） T-lymphocytes proliferation was raised slightly and IDO activity had no change as compared with control group （P〉0. 05）. Conclusion IFN-γ could induce the expression of IDO of MSCs in vitro. IDO could inhibit the T-lymphocyte proliferation.