白细胞介素-35修饰的间充质干细胞诱导免疫耐受的研究

Interleukin-35 gene modified mesenchymal stem cells induced the proliferation of regulatory T cells

目的探讨白细胞介素-35（IL-35）基因修饰小鼠骨髓间充质干细胞（BMSCs）的可行性，并通过体内和体外实验评价其诱导调节性T细胞（Tregs）增殖的能力。方法体外培养的小鼠BMSCs经过鉴定后，采用脂质体转染IL-35表达质粒，采用酶联免疫吸附试验（ELISA）法检测培养上清中IL-35含量，评价IL-35．MSCs分泌IL-35的能力。采用流式细胞方法检测淋巴细胞亚群的变化评价IL-35-MSCs体外混合淋巴细胞培养和静脉注射后诱导Tregs增殖的能力。结果利用全骨髓贴壁法可成功获得小鼠BMSCs，IL-35表达质粒转染后能表达具有生物活性的IL，35。接受静脉注射IL-35-MSCs组小鼠外周CIM＋CD25＋Tregs的比例为（5．24±0．61）％，明显高于注射转染对照质粒的MSCs组的（4．21±0．64）％和单纯MSCs组的（4．32±0．76）％，差异有统计学意义（P〈0．01），而后两者之间差异无统计学意义（P〉0．05）。IL-35-MSCs静脉注射组小鼠脾脏淋巴细胞在单向淋巴细胞混合培养中CIM＋T淋巴细胞和CIM＋CD25＋Tregs比例分别为（1d．47±3．86）％、（4．35±0．57）％，而注射转染对照质粒的MSCs组分别为（20．894-2．32）％、（3．29±0．78）％，单纯MSCs组分别为（18．81±2．8d）％、（3．43±1．02）％，差异有统计学意义（P〈0．05）而后两者之间差异无统计学意义（P〉0．05）。结论IL-35基因修饰的MSCs能促进CD4＋CD25＋Tregs增殖，其作用明显强于转染对照质粒和未转染的MSCs。IL-35和BMSCs发挥协同作用，放大各自的免疫调节和免疫抑制功能。

Objective To investigate the feasibility of interleukin （IL）-35 gene transfection of mice bone marrow mesenchymal stem cells （BMSCs） and the effect of IL-35 gene modified MSCs （IL-35-MSCs） on the the proliferation of regulatory T cells （Tregs）. Methods MSCs were cultured in vitro and transfected with IL-35 expressing plasmid by lipofectamine. The IL-35 expression of IL-35-MSCs was detected by enzyme linked immunosorbent assay （ELISA）. The lymphocytes subsets after one-way mixed lymphocyte culture and in vivo transplantation were analyzed by flow cytometry to evaluate the effect of IL-35-MSCs on the proliferation of Tregs. Results The mice BMSCs can be isolated and cultured by the whole bone marrow adherent method. IL-35 is expressed in the MSCs supernatant and serum after IL-35 in vitro and in vivo transfection. Percentage of CD4＋ CD25 ＋ Tregs in mice treated with IL-35-MSCs increased significantly than control plasmid transfected MSCs and non-transfected MSCs groups（P 〈 0. 01 ）. IL-35- MSCs up-regulated the CIM＋ CD25 ＋ Tregs level in the allogeneic mixed lymphocyte reaction system, low ered the percentage of CD4 ＋ ceils compared with the other two control groups （ P 〈 0. 05 ）. Conclusion IL-35-MSCs enhanced the proliferation of CD4 ＋ CD25 ＋ Tregs in vitro and in vivo compared to control MSCs. IL-35 and MSCs have synergic effects on the negative immune regulation and the induction of im- mune tolerance.