人Th17细胞体外诱导分化条件的研究  

Condition of human Th17 cell differentiation induced in vitro

作　　者：王辰[1] 郭薇[1] 王欣[1] 郁冬梅[1] 王宇恒[1] 姚文兵[1] 

机构地区：[1]中国药科大学生命科学与技术学院,南京210009

出　　处：《中国药科大学学报》2016年第1期106-111,共6页Journal of China Pharmaceutical University

基　　金：国家自然科学基金资助项目(No.81202450;No.81273426);国家教育部博士点基金资助项目(No.20120096110007);江苏省高校研究生科技创新计划资助项目(No.CXZZ13_0331)~~

摘　　要：通过密度梯度离心法从人新鲜血液中分离获得人外周血单核细胞(PBMCs),比较分析加入不同细胞极化因子(IL-1β、IL-6、TGF-β和IL-23)以及诱导不同时间(1,2,3,4 d)对Th17细胞分化的影响,利用流式细胞仪、ELISA方法以及Q-PCR技术考察相关指标,评估人体外Th17细胞的分化水平。研究表明,IL-1β、IL-6、TGF-β和IL-23单独均能对Th17细胞的分化起到促进作用,联用IL-1β、IL-6、TGF-β和IL-23时可使Th17细胞体外分化效率达到最佳。随着诱导时间的延长,Th17细胞分化水平逐渐升高,培养3 d时达到峰值,继续增加培养时间分化率反而出现降低。最终确立人Th17细胞体外分化的最佳诱导条件:在加入CD3抗体和CD28抗体的基础上,同时加入IL-1β、IL-6、TGF-β以及IL-23共同培养人PBMCs3 d可有效地刺激人Th17细胞的分化,并且流式细胞检测Th17细胞分化率达到近10%,ELISA检测细胞上清液中IL-17A含量达到近3 ng/m L,Q-PCR方法分析IL-17 mRNA基因表达升高达15倍,此方案具有操作简便、周期短、成本低廉等优点,为Th17细胞的功能研究及相关疾病治疗药物的研发建立了有效的检测平台。In this study, a series of related indicators were investigated via flow cytometq~, enzyme-linked immu- no-sorbent assay （ELISA） and quantitative real-time PCR （Q-PCR） technology to assess the in vitro differentia- tion of human Thl7 cells. Human peripheral blood mononuclear cells （PBMCs） were purified from fresh human blood using gradient centrifugation and the Thl7 cells were then induced with different eytokines （IL-113, IL-6, TGF-β and 1L-23） at different induction times （ 1,2, 3, 4 d） to compare the effects on Thl7 cell differentiation under these conditions. Data showed that IL-1β, IL-6, TGF-β or IL-23 alone play a promoting role in Thl7 cell differentiation and combination of IL-1β, IL-6, TGF-I3 and IL-23 could induce efficient human Thl7 cell differen- tiation in vitro to achieve the best. Further optimization of the induction time found that the Thl7 cell differentia- tion efficiency gradually increased with the extension of the time; however, when culturing for 3 d, it reached the peak number and then decreased in spite of the time increase. Finally the optimal condition of in vitro polarization of human Thl7 cells was established, in which the purified PBMCs were cultured with anti-CD3 and anti-CD28 as the basal conditions, and co-cultured with IL-113, IL-6, TGF-β and IL-23 for 3 d to effectively induce the differen- tiation of Thl7 cells. The inducing efficiency is significantly higher than that in normal control. At the optimal condition, Th17 cell differentiation frequency （CD4 ＋ IL-17A ＋） was found to increase to nearly 10% through flow cytometry analysis and the secretion level of IL-17A in cell supernatants was also detected to reach 3 ng/mL using ELISA methods. In addition, gene expression of IL-17A was determined by quantitative real-time PCR using pre-designed primers by the comparative method of relative quantitation （AACt） and [3-aetin gene was used as an internal control for sample normalization. The results showed that the expression of IL-17A mRNA coul

关 键 词：人外周血单核细胞 TH17细胞 体外 诱导分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]