摘要
目的观察磁珠分选的LFA-1基因敲除小鼠（LFA-1-/-）CD4＋、CD62L＋naive T细胞在不同刺激因素下向CD4＋Foxp3＋调节性T细胞（iTreg）及CD4＋IL17A＋细胞（TH17）之间转变的可塑性。方法采用LFA-1-/-C57/B6小鼠，免疫磁珠分选小鼠脾脏单个核细胞CD4＋CD62L＋naive T细胞并检测纯度，分选的naive T细胞完全培养基重悬后置于抗体包被的96孔细胞培养板中，分为iTreg组、TH17组（分为加IL6亚组、加IL6及IL23亚组）。其中iTreg组培养体系中添加重组小鼠IL2300U/ml，重组人TGFβ30ng/ml，TH17两亚组分别添加重组小鼠IL630ng/ml，重组人TGFβ3ng/ml及重组小鼠IL2330ng/ml、IL630ng/ml、重组人TGFβ3ng/ml，将96孔培养板置于37℃温箱培养90~108个小时，流式检测Foxp3＋/CD4＋T及IL17A＋/CD4＋T，荧光定量PCR检测分选细胞在不同刺激因素诱导下转录因子Foxp3mRNA及RORγtmRNA表达的变化。结果磁珠分选CD4＋、CD62L＋naiveT细胞纯度高于95%，分选出的naiveT细胞在IL2和TGFβ作用下能诱导出iTreg细胞，其特异性转录因子foxp3mRNA表达升高，而加入TGFβ和IL6或IL23作用下可诱导出TH17细胞，其特异性转录因子RORγtmRNA表达升高。结论 LFA-1-/-小鼠的CD4＋、CD62L＋naive T的诱导分化具有可塑性，在不同刺激因素作用下可实现iTreg和TH17细胞之间的诱导转变。
Objective To explore the plasticity ofiTtreg/TH17 cells induced in vitro by CD4＋CD62L＋ naive T Cells from LFA-1/ mice. Methods CD4＋, CD62L＋ naive T cells from LFA-1-/-/ C57/B6 mice were seperated with MACS and the purity was analyzed by FCM.Naive T cells were divided into two groups, called respectively iTreg group and TH17 group.Cells of the two groups were cultured in 96 volume which were in 4~C incubation of plate-bound anti-CD3mAb and anti-CD28 mAb for one night. Then iTtreg group cells were added with soluble routine IL2 and human TGF β -1 while TH17 group with murine IL6 and/or IL23 together with human TGF β ： 1.All cells were cultured in 37 ~C .The ratio of Foxp3＋/CD4＋T and IL 17A＋/CD4＋T was analyzed by FCM on 60h-108h. The Foxp3 mRNA and ROR 7 t mRNA of cultured cells was measured by qRT-PCR. Results CD/, CD62L＋naive T cells from LFA-1 / mice had differentiated plasticity under different cytokines circumstances
出处
《浙江临床医学》
2013年第6期769-772,共4页
Zhejiang Clinical Medical Journal
基金
国家自然科学基金面上项目资助（编号：81170359）
