体外培养扩增CD4^＋CD25^＋调节性T细胞的实验观察  

Proliferation of CD4^＋ CD25^＋ regulatory T cells of rat by different cytokines in vitro

作　　者：王子函[1] 朱继业[1] 李涛[1] 冷希圣[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

出　　处：《中华医学杂志》2008年第12期844-847,共4页National Medical Journal of China

基　　金：国家自然科学基金资助项目（30772050）

摘　　要：目的观察细胞因子体外培养扩增CD4^＋CD25^＋调节性T细胞（Treg）的作用，以便从体外获取大量Treg细胞。方法将从C57BL/6幼稚小鼠脾脏和淋巴结中提取的Treg与从DBA／2小鼠骨髓中提取的成熟树突细胞（mDC）混合培养，并分别加入白细胞介素-2（IL-2）、IL-4或IL-15，检测Treg增殖和凋亡的情况，与不加入任何细胞因子为对照。分别将培养获得的Treg与C57BL/6幼稚小鼠的效应性T细胞（Teff）混合培养，测定扩增后的Treg对Teff的抑制活性。检测扩增后Treg的Foxp3表达，从而证明培养获得的Treg仍保持其表型。结果在保持其对Teff抑制活性的同时，IL-2组、IL-4组、IL-15组Treg的增殖细胞前体频率分别为31．3％、28．9％、34．5％，明显高于对照组（14．5％），均P〈0．05；增殖指数分别为1．9、1．7、1．8，明显高于对照组（1．5），均P〈0．05。IL-2组、IL-4组、IL-15组Treg的凋亡细胞比例分别为12．8％、11．4％、12．7％，均明显低于对照组（28．9％），均P〈0．05。扩增后的Treg仍高表达Foxp3（91．75％）。结论IL-4、IL-15和IL-2一样，具有促进Treg增殖、减少其凋亡的作用，并同时保持对Teff的抑制功能。扩增后的Treg仍保持其表型，高表达Foxp3。Objective To evaluate the effects of cytokines on the proliferation and function of CD4^＋CD25^＋ regulatory T cell （Treg）. Methods Tregs were isolated from naive C57BL/6 mice spleen and lymph nodes. Mature dendritic ceils （mDC） were isolated from DBA/2 mice, co-cultured with Tregs, and divided into 4 groups with or without interleukin-2 （ IL-2）, interleukin-4 （ IL-4）, and interleukin-15 （ IL- 15） added into the culture fluid. Fluorescence-activated cell sorting （FACS） was used to detect the Treg proliferation and apoptosis with CFSE and annexin-V staining. The co-culture increased Tregs were divided into 5 groups： CFSE labeled naive CD4^＋CD25^- T cells, self-proliferated Treg, Treg mixedly cultured with IL-2 mDC, and Teff, Treg mixedly cultured with IL-4, mDC, and Teff, and Treg mixedly cultured with IL- 15, mDC, and Teff, a control group included Teff co-cultured with mDC. FACS was used 5 d later to evaluate the suppressive function of the Treg on the Teff. The expression of Foxp3, indicating the phenotype of Treg was detected. Results FASC showed that the values of precursor frequency （PF） of the Tregs stimulated by IL-2, IL-4, and IL-15 were 31.3%, 28.9%, and 34.5% respectively, all significantly higher than that of the control group （ 14.5% all P 〈 0.05 ）, and the values of proliferation index （PI） of the Tregs stimulated by IL-2, IL-4, and IL-15 were 1.9, 1.7, and 1.8 respectively, all significantly higher than that of the control group （ 1.5, all P 〈 0.05）. The apoptotic rates of the Tregs stimulated by IL-2, IL- 4, and IL-15 were 12.8%, 11.4%, and 12.7% respectively, all significantly lower than that of the control group （28.9%, P〈0.05）. The Foxp3 expression rate of the Tregs stimulated by IL-2, IL-4, and IL-15 was 91.75%. Conclusion IL-2, IL-4, and IL-15 in the in vitro culture of Treg stimulate the Treg proliferation, reduce their apeptosis, and maintain their suppressive function. The proliferated Tregs still maintain their phenotype, highly expre

关 键 词：T淋巴细胞 白细胞介素类 调节性T细胞 细胞培养 

分 类 号：R686[医药卫生—骨科学]