不同培养环境对PMBC中Treg和Th17细胞分化的影响

Effect of different culture conditions on differentiation of Treg and Th17 in PBMC

目的:研究不同培养环境对调节性T细胞(regulatory T cells,Treg)和Th17细胞分化的影响,探索能够逆转肿瘤微环境诱导的免疫耐受的手段。方法:克隆人白介素-6(IL-6)的基因,将其转入表达TGF-β的肿瘤细胞株中,并筛选稳转系;分别收集含或不含IL-6的肿瘤细胞培养上清,制备成条件培养基(Conditioned medium,CM),用于体外培养人外周血单核细胞(Peripheral blood mononuclear cells,PBMC),流式检测PBMC在不同培养环境中Treg和Th17细胞的变化情况。结果:RTPCR分析表明BEL-7402表达TGF-β的水平明显高于Hep G2,因此选择BEL-7402用于制备IL-6的稳转系;ELISA检测结果表明所制备的稳转系能够有效地表达IL-6;流式结果表明,由肿瘤细胞培养上清制备的CM能明显诱导T细胞分化为Treg,而在IL-6存在的情况下则能明显抑制Treg细胞的形成,并促进T细胞分化为Th17细胞。结论:肿瘤细胞培养上清促使PBMC中Treg比例增加;而在其中添加IL-6则能够降低Treg细胞比例,并增加Th17细胞的比例。

Objective：To investigate the effect of different culture conditions on the differentiation of Treg and Th17 to lay a foundation for exploring the methods to reverse the immune tolerance induced by tumor microenvironment. Methods： The IL-6 gene was cloned and stablely transferred into the tumor cell line expressing TGF-β. The conditioned mediums（ CM） were prepared by collecting the culture supernatants of tumor cell lines with or without IL-6 expression and used in the in vitro culture of peripheral blood mononuclear cells（ PBMC）. The changes of Treg and Th17 in PBMC treated with different CM were detected with flow cytometry（ FCM）. Results： The expression of TGF-β in BEL-7402 was higher than that in Hep G2. Thus the BEL-7402 was selected for preparation of cell line stablely transfected with IL-6 gene. ELISA detection confirmed the effective expression of IL-6 by the identified cell lines. It was showed that the Treg increased in PBMC treated with culture supernatants of tumor cells. However,the presence of IL-6 reversed the increase of Treg and promoted the differentiation of Th17. Conclusion： The culture supernatants of tumor cells increases the proportion of Treg. However,the presence of IL-6 in this CM can reverse the increase of Treg and raise the proportion of Th17.