不同培养方法对体外诱导分化Th17细胞的影响  

Different culture methods to induce Th17 cell differentiate in vitro

作　　者：张新[1] 费先艳 于成功[1] 

机构地区：[1]南京大学医学院附属鼓楼医院消化科,江苏南京210008

出　　处：《南京医科大学学报（自然科学版）》2016年第3期335-339,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(81170359)

摘　　要：目的 :Th17细胞缺乏特异性表面标记,难以分离纯化活细胞,通过调整细胞因子浓度和不同培养方法 ,以获得高纯度Th17细胞亚群。方法:选用C57BL/6J小鼠脾脏获得单个核细胞,磁珠分选获得CD4+、CD44-Naive T细胞。建立不同Th17诱导培养体系,通过流式细胞术检测获得Th17细胞的比例,荧光定量PCR方法检测ROR-γt、IL-17 m RNA表达水平,ELISA方法检测细胞培养上清中IL-17浓度,以寻找最佳培养条件。结果:TGF-β+IL-6+IL-23可诱导Th17细胞,但比例较低;加入TNF-α、IL-1β、anti-IFN-γ、anti-IL-2可进一步提高Th17比例,但后期培养呈衰减趋势;使用U型96孔板培养Th17细胞明显优于24孔板培养,细胞分化呈稳定上升趋势,培养第9天Th17细胞可达(91.85±1.05)%。IL-17、ROR-γt m RNA与Naive T细胞相比明显上调(P<0.001),IFN-γ、IL-4、Foxp3 m RNA表达量低(P<0.001),细胞培养上清液中IL-17浓度明显升高(P<0.001)。结论:调整细胞因子诱导方案、缩小培养面积可不同程度提高Th17细胞诱导比率,且培养面积的影响作用更为显著。Objective：As Th17 cells lack of specific surface marker,it is difficult to achieve highly purified population of Th17 cells. We sought to obtain highly purified population of Th17 cells by adjusting the concentration of cytokines and different cultivation methods. Methods：CD4＋CD44-naive T cells were separated from C57 BL / 6J mice by magnetic-activated cell sorting（MACS）.Different inducing systems were used for testing the effect of different cytokines and culture methods. The ratio of Th17 cells was analyzed by flow cytometry,the expressions of IL-17,ROR-γt,IFN-γ,IL-4 and Fox P3 m RNA were measured by q RT-PCR method and the IL-17 concentration in cell culture supernatant was detected by ELISA. Results：Th17 cells were induced by TGF-β,IL-6 and IL-23,the ratio of Th17 increased by adding TNF-α,IL-1β,anti-IFN-γ and anti-IL-2. The use of U-type 96 wells plate was significantly better than the 24 wells plate in culturing Th17 cells. Th17 cells showed steady growth when cultured in U-type 96 wells plate and the ratio of Th17 cells was significantly increased[（91.85 ± 1.05）%]. The expressions of IL-17 and ROR-γt m RNA were up-regulated compared with that of Naive T cells（P〈0.001）,however,IFN-γ,IL-4 and Foxp3 m RNA were decreased（P〈0.001）. The concentration of IL-17 in cell culture supernatant was significantly increased（P〈0.001）. Conclusion：The regulated method can generate a highly purified population（90%） of Th17 cells.

关 键 词：TH17细胞 NAIVE T细胞 体外诱导 细胞因子类 

分 类 号：R392.3[医药卫生—免疫学]