CD4^＋CD25^-T淋巴细胞转化为CD4^-CD8^-调节T细胞的体外实验研究  

In vitro conversion of CD4 ＋ CD25- T cells to CD4- CD8- regulatory T cells

作　　者：苏万春[1] 冷希圣[1] 张栋[2] 高鹏骥[1] 朱继业[1] 

机构地区：[1]北京大学人民医院肝胆外科中心北京大学器官移植中心,100044 [2]首都医科大学附属北京友谊医院实验中心

出　　处：《中华普通外科杂志》2012年第6期479-482,共4页Chinese Journal of General Surgery

基　　金：基金项目：国家自然科学基金科学部主任基金资助项目（30940071）

摘　　要：目的探讨体外诱导和纯化CD4^＋CD25^-T淋巴细胞（effector T cell，Teff）转化为CD4^＋CD8^-双阴性调节T细胞（double negative regulatory T cell，DNTreg）的最适条件。方法采用免疫磁珠分选方法提取C57BL／6小鼠的CD4^＋CD25^-T淋巴细胞、DBA／2小鼠的成熟树突状细胞共培养，加入不同剂量的IL-2，通过流式细胞检测CD4^-CD8^-T细胞的转化比例并确定最适条件，免疫磁珠阴性选择分选提纯转化的CD3^＋CD4^-CD8^-T细胞，流式细胞仪检测转化的CD4^-CD8^-调节T细胞对CD4^＋CD25^-效应T细胞增殖抑制情况。结果CD4^＋CD25^-T淋巴细胞与DBA／2小鼠的树突状细胞共培养6d后检测Cd4^-CD8^-调节T细胞的转化比率为6．21％±2．03％，实验组加入不同浓度IL-2的转化率：A组（25ng／ml）为14．77％±2．15％，B组（50ng／m1）为21．29％±2．68％，C组（75ng／m1）为43．45％±4．45％，D组（100ng／ml）为28．59％±3．05％，IL-2浓度在75ng／ml时，转化获得率最高（C组与对照组、实验A、B、D组比较分别t=10．700，8．288，6．158，3．932，均P〈0．05）；分离提纯CD4^-CD8^-双阴性调节细胞纯度达到98．10％，CD4^-CD8^-双阴性调节细胞与CFSE染色的CIM^＋CD25^-T淋巴细胞、小鼠树突状细胞共培养6d，实验组增殖指数为1．15明显低于对照组2．07。结论小鼠CD4^＋CD25^-T淋巴细胞在体外，成熟树突状细胞刺激下可转化为CD4^-CD8^-双阴性调节T细胞，IL-2可显著提高其转化率。Objective To optimize the condition of converting murine naive CD4 ＋ CD25 - T cells （effeetor T cells, Teffs） to CD4- CD8- double negative regulatory T cells （DN Tregs） in vitro. Methods Naive Teffs from C57BL/6 mouse were isolated with magnetic activated cell sorting（MACS） and co-cultured with DBA/2 mature dendritic cells （mDCs） with different doses of recombinant murine interleukin-2 （IL-2）. The percentage of converted DN Tregs was examined by flow eytometry after 6 days. Purified DN Tregs were co-cultured with CFSE labeled Teffs. The proliferation rate of Teffs were evaluated by flow cytometry. Results Without IL-2, the percentage of CD4- CD8- T cells was 6. 21% ± 2. 03%. With IL-2, the percentage was 14.77% ±2. 15% （25 ng/ml）, 21.29% ±2. 68% （50 ng/ml）, 43.45% ± 4. 45% （75 ng/ml）, and 28. 59% ±3.05% （ 100 ng/ml） respectively. The IL-2 concentration of 75 ng,/ml significantly enhanced the conversion of Teffs to DN Tregs （separately t = 10. 700,8. 288,6. 158,3. 932, all P 〈 0. 05 ）. Highly purified DN Tregs significantly suppress the proliferation of Teffs in vitro. Conclusions Teffs are converted to DN Tregs in vitro with the LPS-aetivated allogeneie mDCs and that 75 ng/ml of IL-2 is the ootimal concentration for the conversion of Teffs to DN Tregs in vitro.

关 键 词：T淋巴细胞 调节性 树突细胞 细胞培养技术 

分 类 号：R392[医药卫生—免疫学]