下调Foxp3基因表达对人调节性T细胞功能的影响  

Effects of Foxp3 knockdown on the functions of human regulatory T cells

作　　者：孙磊[1] 易寿南[1] 陈丽[1] 

机构地区：[1]山东大学齐鲁医院内分泌科,济南250012

出　　处：《中华医学杂志》2011年第30期2124-2128,共5页National Medical Journal of China

摘　　要：目的利用RNA干扰技术下调Foxp3表达，观察其对人CD4^＋CD25^＋调节性T细胞（Treg）表型及功能的影响。方法利用Foxp3siRNA转染人CD4^＋CD25^＋Treg，荧光显微镜和流式细胞仪（FACS）检测转染效率，实时定量PCR和Western印迹分别鉴定Foxp3基因及Treg功能相关基因表达、Foxp3蛋白表达，细胞增殖实验检测Treg抑制功能，FACS测Treg表型标记，ELISA检测细胞因子表达。结果Foxp3siRNA转染人CD4^＋CD25^＋Treg效率为68％，与对照组相比，转染后的细胞Foxp3基因下调了61．4％（P〈0．01），Foxp3蛋白表达较低，Treg功能相关基因和主要表型标记也较对照组有改变，差异有统计学意义。功能试验提示无论是在异种还是同种抗原刺激的淋巴细胞反应中，Foxp3 siRNA转染的Treg与对照组相比，对效应性T细胞的抑制作用较弱（异种抗原组83％比48％，P〈0．05；同种抗原组65％比28％，P〈0．01），且主要抑制性和效应性细胞因子的表达下降，差异有统计学意义。结论Foxp3是人天然Treg维持其抑制性功能的核心细胞内标志。Objective To employ the technology of interfering RNA （RNAi） to identify the role of Foxp3 in the in vitro suppressive effect of human regulatory T cell （Treg） on effector T cells. Methods Expanded human Treg were transfeeted with siRNA targeting Foxp3 genes. The transfection efficiency, the level of corresponding gene and its protein expression were measured by fluorescent microscopy, fluorescence-activated cell sorting （FACS）, real-time PCR and Western blot respectively. The phenotypes of Treg were analyzed by FACS. The aiRNA transfeeted Treg was then co-cultured with porcine PBMC or human PBMC-stimulated autologuus CD4 ^＋ CD25 - T cells. Their proliferations were examined by WST-1. Treg and autologuus CD4 ＋ CD25 - T cell-related suppressive cytokines were assessed by ELISA. Results A 68% transfeetion efficiency in expanded Treg was achieved for Foxp3 siRNA. Real-time PCR revealed a 61.4% mRNA knockdown induced by siRNA targeting Foxp3 genes in Trog versus the control （P 〈0.01 ）. Some Treg-associated surface markers were significantly altered versus the control. And the production of suppressive eytokines was lowered. These changes were correlated with the diminished Treg activity in suppressing the proliferation of effeetor CD4 ＋ CD25 - T cells. There was 83% suppression by non- transfected Treg vs 48% suppression by Foxp3 siRNA transfected Treg in xeno-immune response （ P 〈 0.05 ） ; and 65% suppression by non-transfected Treg vs 48% suppression by Foxp3 siRNA transfeeted Treg in al]o-immune response （ P 〈 0. 01 ）. Conclusion Foxp3 is a key intracellular marker for maintaining the phenotypes and functions of Treg.

关 键 词：叉头转录因子类 RNA 小分子干扰 T淋巴细胞 调节性 

分 类 号：R3[医药卫生—基础医学]