结核菌素纯蛋白衍生物刺激人γδT细胞效应分析  

Activation effect of tuberculin pure protein derivative on γδT cell

作　　者：黄方[1] 杜先智[1] 

机构地区：[1]重庆医科大学附属第二医院呼吸内科,重庆400010

出　　处：《第三军医大学学报》2011年第5期481-484,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30872261)~~

摘　　要：目的探讨结核菌素纯蛋白衍生物(pure protein derivative,PPD)对人外周血γδT细胞的刺激效应。方法采用Ficoll密度梯度离心法分离人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)后,经尼龙毛柱法分离得T细胞。分别以卡介苗和PPD刺激γδT细胞,流式细胞仪分选纯化γδT细胞,并检测其抗原提呈细胞表型。按照T细胞培养基成分将实验分为4组(n=20):①空白组:结核分枝杆菌分泌性抗原(Mtb-Sag)25μg/ml+CFSE标记的T淋巴细胞;②卡介苗(BCG)组:BCG刺激γδT细胞+Mtb-Sag 25μg/ml+CFSE标记T淋巴细胞;③PPD组:PPD刺激γδT细胞+Mtb-Sag 25μg/ml+CFSE标记T淋巴细胞;④树突状细胞(dendritic cell,DC)组:成熟DC+Mtb-Sag 25μg/ml+CFSE标记T淋巴细胞。9 d后检测T细胞及CD4+T细胞的增殖情况。结果与静止期相比,PPD可刺激γδT细胞CD80、CD86和HLA-DR表达水平升高至(39.07±1.26)%、(51.93±1.85)%和(45.74±1.58)%(P<0.05),与BCG刺激效应相似[(42.12±1.72)%,(54.67±2.01)%,(49.79±1.61)%,P>0.05]。PPD组T细胞总数(5.14±0.68)×106和CD4+T细胞所占比例(35.01%)较空白组显著升高(P<0.05),与BCG组[(4.6±0.77)×106,32.00%)]相似,显著低于成熟DC细胞组[(7.50±0.35)×106,58.94%](P<0.05)。结论 PPD可激活γδT细胞CD80、CD86和HLA-DR表达,提示PPD可能具有激活人外周γδT细胞功能。Objective To explore the activation effect of tuberculin pure protein derivative（PPD） on γδT cells of human peripheral blood.Methods Human peripheral blood mononuclear cells were separated with Ficoll density gradient centrifugation method,and T cells were separated with a nylon fiber column.After separately stimulated with bacillus calmette-guerin（BCG） and PPD,the γδT cells were sorted and purified with a flow cytometer,and their antigen-presenting cell phenotypes were detected.According to culture medium components of T cells,four experimental groups were established,i.e.,（1） blank group： Mycobacterium tuberculosis（Mtb） secretory antigen（Mtb-Sag） 25 μg/ml＋CFSE-labeled T cells,（2） BCG group： BCG-stimulated γδT cells ＋ Mtb-Sag 25 μg/ml＋CFSE-labeled T cells,（3） PPD group： PPD-stimulated γδT cells＋Mtb-Sag 25 μg/ml＋CFSE-labeled T cells,and（4） dendritic cell（DC） group： mature DC＋Mtb-Sag 25 μg/ml＋CFSE-labeled T cells.The proliferation of T cells and CD4-positive T cells was detected.Results Compared with the resting phase,PPD could stimulate the expression of CD80,CD86 and HLA-DR of γδT cells to reach levels of 39.07±1.26%,51.93±1.85% and 45.74±1.58%（P0.05）,respectively,demonstrating effects comparable to BCG stimulation [（42.12±1.72）%,（54.67±2.01）% and（49.79±1.61）%,P0.05,respectively].The T cell number [（5.14±0.68）×106] and the CD4-positive T cell ratio（35.01%） in the PPD group were remarkably higher than those in the blank group [（1.22±0.02）×106,8.28%,P0.05],similar to those in the BCG group [（4.6±0.77）×106,32.00%],and remarkably lower than those in the DC group [（7.50±0.35）×106,58.94%,P0.05].Conclusion PPD can stimulate the expression of CD80,CD86 and HLA-DR of γδT cells,indicating its activation effects on human peripheral blood γδT cells.

关 键 词：结核菌素纯蛋白衍生物 ΓΔT细胞 流式细胞术 

分 类 号：R378.911[医药卫生—病原生物学] R392.11[医药卫生—基础医学]