摘要
目的探讨IL-15抑制CD4+CD25+调节性T细胞(Tregs)凋亡的作用及机制,为临床应用IL-15提供理论依据。方法采用免疫磁性细胞分离法分离人外周血中的Tregs,流式细胞仪检测该细胞纯度。将Tregs细胞培养液中加入Dynabeads CD3/CD28T cell expander,依据是否加入细胞因子分为三组:对照组(不加入任何细胞因子);IL-2组(100U/ml)和IL-15组(50ng/ml)。流式细胞仪检测细胞凋亡率,3H-TdR掺入法检测细胞因子存在下Tregs抑制CD4+CD25-效应T细胞(Teff)增殖的免疫功能,Western-blot法检测细胞内Bcl-2、Bcl-xl及p-Bad(ser112)的变化。结果分选后Tregs纯度为95.2%,胞内因子染色Foxp3在Tregs中的表达率为94.2%。IL-2组和IL-15组的细胞凋亡率(即凋亡细胞与细胞总数之比)分别为(11.32±0.43)%和(9.89±5.38)%,二者之间无显著性差异,但均显著低于对照组(87.12±1.43)%,P<0.001。在细胞因子存在的情况下,IL-2组活化的Teff增殖较对照组和IL-15组显著性强,P<0.001;此时Tregs抑制Teff增殖的功能较对照组和IL-15组下降,P<0.001;而在IL-15存在的情况下,IL-15组活化的Teff增殖与对照组比较,无显著性差异,P>0.05,并且IL-15组Tregs对Teff增殖的抑制作用与对照组比较无显著性差异,P>0.05。Western-Blot显示三组TregsBcl-2表达水平相当;但是IL-2组和IL-15组的Bcl-xl和p-Bad(ser112)表达较对照组显著性上调。结论 IL-15能抑制Tregs凋亡,其作用与IL-2相当,其机制可能是通过上调Bcl-xl的表达和Bad(ser112)的磷酸化,而且IL-15存在的情况下,Tregs抑制功能明显比IL-2存在时强。
The aim of our study is to investigate the effects of IL-15 on the apoptosis of CD4＋CD25＋ regulatory T cells （Tregs） and the mechanism. We isolated Tregs from the human peripheral blood mononuclear cells in two steps by magnetic cell sorting system, and measured the purity rate of the sorted cells was by flow cytometry （FC）. Then the prepared Tregs were co-cultured with Dynabeads CD3/CD28 T cell expander and divided into 3 groups （control group, IL-2 group, and IL-15 group） without or with IL-2 （100 U/ml） and IL-15 （50 ng/ml）. FC was used to detect the Tregs apoptosis; the incorporation of [3H] thymidine was used to evaluate the suppressive function of the Tregs on the Teffin the presence of IL-2 or IL-15; Western blotting was used to observe the expression of Bcl-2, Bcl-xl, and p-Bad（serH2）. FC showed that the purity of sorted CD4＋CD25＋ T cells was 95.2 %; the intracellular staining indicated that the expression rate of Foxp3 was 94.2% in Tregs. Compared with Tregs cultured without cytokines, IL-15 and IL-2 were able to promote survival of activated Tregs. IL-2 profoundly reversed the Tregs-mediated suppression of Teff proliferation, however IL-15 had almost no effects on Tregs-mediated suppression. We also found that addition of IL-15 and IL-2 did not affect Bcl-2 expression. In contrast, Bcl-xl and p-Bad （serH2） was selectively up-regulated by IL-15 and IL-2 in Tregs. All the results indicated that IL-15 can reduce the Tregs apoptosis, which maybe associated with up-regulating the Bcl-xl and p-Bad（ser12） expression. Furthermore, IL-15 could maintain Tregs suppression function.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2010年第3期224-227,231,共5页
Immunological Journal
基金
国家自然科学基金(30772048)
重庆市自然科学基金(CSTC2006BB5066)
贵州省优秀科技教育人才省长资金项目[黔省专合字(2005)230
(2009)31]
西南医院临床创新基金(SWH2006A003)
