体外培养扩增CD4+CD25+调节性T细胞的实验观察

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.

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

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 cells (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 na?ve 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 apoptosis, and maintain their suppressive function. The proliferated Tregs still maintain their phenotype, highly expressing Foxp3.