IFN-γ经共抑制分子B7-H1正向调控小鼠骨髓间充质干细胞免疫抑制的研究  

Mouse bone marrow derived mesenchymal stem cells suppress lymphocyte proliferation through coinhibitor B7-H1 upregulated by IFN-γ

作　　者：盛慧明[1] 黄红宇[1] 魏云玉[1] 王瑛[2] 王利[3] 沈佰华[3] 李宁丽[3] 

机构地区：[1]南京医科大学附属无锡二院中心实验室,214002 [2]上海中医药大学附属岳阳中西医结合医院检验科 [3]上海交通大学医学院,上海市免疫学研究所,200025

出　　处：《中华微生物学和免疫学杂志》2009年第4期345-350,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家重点基础研究发展计划973项目（2005CB522705-3）

摘　　要：目的探讨IFN-γ通过B7-H1／PD-1通路正向调节骨髓间充质干细胞（MSC）免疫抑制作用机制，为MSC用于治疗免疫性疾病提供新的理论依据。方法首先体外获得纯化鼠源MSC并进行三向分化鉴定；间充质干细胞和淋巴细胞反应体系进行共培养，^3H掺入试验测定细胞增殖水平；ELISA检测共培养上清中IFN-γ、TGF—β、TNF-α、IL-10因子的表达水平；流式检测共培养中MSCB7-H1分子的变化；siRNA干扰MS CB7-H1后，^3H掺入试验检测淋巴细胞增殖水平的改变，采用Transwell培养方法确认MSC通过细胞间接触发挥作用。结果体外鼠源MSC经过5代的反复贴壁纯化之后，形态均一纯度高，并具有向脂肪细胞、骨细胞和软骨细胞分化的能力；MSC与混合淋巴细胞反应、ConA刺激的淋巴细胞反应以及anti—CD3／CD28刺激活化的淋巴细胞反应混合共培养3H掺人试验结果均显示增殖反应被抑制（P=0．0167，0．0081，〈0．0001），其抑制作用呈现对MSC细胞的剂量依赖；共培养上清中细胞因子IFN-γ、TNF—α均显著升高，而TGF—β、IL-10未检测到，同时共培养中MSC表面B7-H1分子明显表达上调（P〈0．05）；经B7-H1特异siRNA干扰MSC后，共培养并检测淋巴细胞增殖反应，细胞增殖水平明显较干扰前提高（P〈0．05），Transwell膜分隔后MSC抑制T细胞活化明显减弱（P〈0．05）。结论MSC经共培养环境中IFN-γ刺激后，表面B7-H1分子的表达上调并通过B7-H1／PD-1通路抑制T细胞活化增殖。将来这种MSC可以用于治疗多种自身免疫性疾病。Objective To study the mechanism of mesenchymal stem cells immunosupression lymphocyte proliferation via B7-H1/PD-1 pathway upregulated by IFN-γ. Methods Bone marrow derived mesencbymal stem cells （MSC） were isolated and purified by repeat adherent passage and detected them multipotential differentiation in conditioned culture medium. Then MSC were cocuhured with lymphocyte proliferation and assayed the level using 3H-thymidine incorporation. Meanwhile, ELISA measured IFN-γ, TGF-β, TNF-α and IL-10 in the cocuhured supernatant and analyzed variation of B7-H1 molecular profile in MSC by flow eytometry. At last siRNA technology was deploied to interfere B7-H1 expression and analyzed MSC immunosuppression on lymphocyte proliferation. Results In vitro the isolated MSC become homogeneous spidie-shaped adherent cells after five passages, and in conditioned culture medium they could differentiate into adipocytes, osteocytes and chondrocytes. In the cocuhure of MSC with mixed lymphocyte, lymphocyte proliferation stimulated by Con A or by anti-CD3/CD28 antibody. Tbe cpm value of the proliferation detected by 3H thymidine incorporation showed MSC suppressed the proliferation significantly （P=0. 0167, 0. 0081, 〈 0.0001 ） and the suppressive potential in a dose-dependent fashion. In the cocuhured supernatant cytokine IFN-γand TNF-α were detected in high concentration, but TGF-β, IL-10 were undetected. Simultaneously MSC in the cocuhure upregnlated B7-H1 expression from basic expression 7% to higher than 70% （P 〈 0.05 ）. After interfere B7-HI expression in MSC by specific siRNA, we detected lymphocyte proliferation and got higher cpm by 3H thymidine incorporation（P 〈 0.05）. Conclusion MSC upregulated B7-H1 molecular expression upon the stimuli of IFN-γ, and through the B7-H1/PD-1 pathway mediated immunosupression on lymphocyte proliferation.

关 键 词：骨髓间充质干细胞 免疫抑制 B7-H1 干扰素Γ 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]