第三方骨髓间充质干细胞诱导同种异体移植受体免疫耐受机制的研究  

作　　者：齐丙迪[1] 孟宝玺[1] 杨阳[2] 刘蓓[2] 李翅翅[2] 夏炜[2] 郭树忠[2] 张晨[3] 

机构地区：[1]遵义医学院,遵义563003 [2]医大学西京医院全军整形外科研究所 [3]大连大学附属新华医院整形外科

出　　处：《中华整形外科杂志》2011年第3期207-212,共6页Chinese Journal of Plastic Surgery

基　　金：辽宁省教育厅高校科技攻关基金资助项目（05L019）

摘　　要：目的初步研究第三方骨髓间充质于细胞（bone marrow-derived mesenchymal stemcells，BMSCs）诱导同种异体移植受体免疫耐受的作用机制。方法40只雌性C57BL／6小鼠作为供体，40只雄性BALB／C小鼠作为受体，建立稳定的同种异体皮肤移植模型，BMSCs取自sD大鼠骨髓。将40只BALB／C小鼠随机分为4组，每组10只。①空白对照组：只进行皮肤移植，未给予其他治疗；②环磷酰胺组（CP组）：大剂量环磷酰胺（cyclophosphamide，CP）腹腔注射，200mg／kg，连用2d（q．d．）；③单纯给予SD．BMSCs移植组（SD-BMSCs组）：移植当天自受体小鼠尾静脉输注2×10。个SD．BMSCs；④细胞药物联合应用组（CP＋SD-BMSCs组）：大剂量CP腹腔注射，200mg／kg，连用2d（q．d．），并于移植当天自受体小鼠尾静脉输注2×10。个SD-BMSCs。检测指标包括：移植皮片存活情况；SD大鼠BMSCs表面抗原CD29、CD34、CD45和CD90鉴定；流式细胞仪检测受体脾脏调节性T细胞（CD4^＋、CD25^＋、Foxp3^＋、Treg细胞）的比例；ELISA检测受体外周血TGF-p、IL-10、IFN-1的含量；异基因T淋巴细胞与经60^Co照射的不同来源BMSCs共培养后，MTT法测定异基因T淋巴细胞增殖的情况。结果CP＋SD-BMSCs组皮肤移植物存活时间为（15．7±1．4）d，空白对照组为（6．1±1．1）d，CP组为（12．3±1．5）d，SD-BMSCs组为（12．6±1．8）d，CP＋SD—BMSCs组皮肤移植物存活时间明显比后3组延长（P〈0．05）。全骨髓贴壁培养的BMSCs表面抗原鉴定：CD29^＋、CD44^＋分别为99．7％和96．7％，CD34-、CD45-分别为1．6％和1．3％。流式细胞仪检测Treg含量SD-BMSCs组和CP＋SD-BMSCs组明显高于空白对照组和CP组（P〈0．05）；ELISA检测受体外周血SD-BMSCs组和cP组TGF—B和IL-10明显高于空白对照组，SD—BMSCs和cP组IFN-γ则明显低于空白对照组（P〈0．05）；共培养结果显示：来源于C57小鼠和sD大鼠的BMObjective To study the immuno-tolerance mechanism of the third-party bone marrowderived mesenchymal stem cells ( BMSCs) in the allogeneic transplantation. Methods Forty female C57BL/ 6 mice and forty male BALB/C mice were respectively used as donors and recipients in skin allogenic graft model. Forty male BALB/C mice were divided randomly into 4 groups: blank control group, CP group, BMSCs group , CP + BMSCs group , with 10 mice in each group. Before skin graft, high-dose abdominal injection of cyclophosphamide ( 200 mg/kg,2 d,q. d. ) was performed in recipient mice in CP and CP + BMSCs groups. On the transplantation day, a bonus of 2 x 106 BMSCs from the SD rat (SD-BMSCs) were injected through the tail vein in the BMSCs and CP + BMSCs groups. The observation and HE staining of skin grafts were used. The expressions of CD29, CD34, CD45 and CD90 of cells were analyzed by using flow cytometry in order to identify BMSCs. The CD4+ , CD25+ , Foxp3+ and Treg cells of spleen were detected by flow cytometry. Cytokine in peripheral blood of recipient mice were measured by ELISA,including TGF-β, IL-10 and IFN-γ. T cells were co-cultured with 60 Co-irradiated bone marrow MSCs from different individuals. The proliferative activity of T cells were evaluated with MTT assay. Results The skin graft survival time was significantly prolonged in the CP + BMSCs group, as compared with that in the blank control group, the CP group, the BMSCs group, respectively. Cells cultured by whole bone marrow adherent cultivation showed CD29+ (99.7% ) ,CD44+ (96.7% ) ,CD34 (1.6% ) ,CD45( 1. 3% ). Compared with the control group and CP group, the ratio of the CD4+ ,CD25+ ,Foxp3+ and Treg cells significantly increased in the SD-BMSCs group and CP + BMSCs group (P ＜ 0. 05). Analysis of peripheral blood by ELISA showed significant high level of TGF-β, IL-10 and low level of IFN-γ in BMSCs group and CP group, compared with that in control group. When co-cultured with BMSCs from different individuals, T- lymphocytes proliferation decreased appar

关 键 词：骨髓间充质干细胞 同种移植 皮肤移植 免疫耐受 

分 类 号：R392[医药卫生—免疫学]