多色流式细胞术检测CHB患者外周血多重细胞因子分泌的Th细胞亚群  

Measurement of multiple cytokine producing T helper cells in the peripheral blood of patients with chronic hepatitis B by multi-color flow cytometry

作　　者：姜钰[1,2] 王蓓蓓 孔雅娴[1,2] 李玉鑫 汪笛[1,2] 宋洋子 王宪波 曾辉 

机构地区：[1]首都医科大学附属北京地坛医院传染病研究所,北京100015 [2]新发突发传染病研究北京市重点实验室,北京100015 [3]首都医科大学附属北京地坛医院中西医结合中心,北京100015

出　　处：《中国肝脏病杂志（电子版）》2017年第4期43-48,共6页Chinese Journal of Liver Diseases：Electronic Version

基　　金：国家重点研发计划(2016YFC1200805);首都临床特色应用研究(Z171100001017052)

摘　　要：目的建立多色流式胞内细胞因子检测方法,明确慢性乙型肝炎(chronic hepatitis B,CHB)免疫清除期患者外周血Th淋巴细胞亚群细胞因子的分泌特点,探讨CHB患者多重细胞因子的表达模式。方法选取2017年3月至2017年4月就诊于首都医科大学附属北京地坛医院的CHB免疫清除期患者10例及健康对照者10例为研究对象。根据多色流式荧光搭配原则,确立外周血T淋巴细胞CD3、CD4、CD8及细胞因子GM-CSF、TNF-α、IFN-γ的六色流式细胞配色方案。利用健康对照者外周血单个核细胞(peripheral blood mo-nonuclear cell,PBMC)调节最适检测电压及荧光补偿,明确PMAIonomycin的体外刺激方案。分离10例CHB患者外周血PBMC细胞,PMA-Ionomycin体外刺激5小时后,行多重流式细胞因子染色,利用多色流式细胞仪LSR Fortessa获取细胞,Flowjo10.0流式细胞软件分析健康对照者及CHB患者外周血Th细胞亚群胞内细胞因子共表达水平。采用Graph Pad Prism 7.0对所得数据进行统计分析。结果建立了多重细胞因子的胞内染色方案,明确了GM-CSF、TNF-α、IFN-γ在Th细胞上的共表达分析策略,确认存在分泌双重细胞因子GM-CSF^+TNF-α^+、GM-CSF^+IFN-γ^+的Th细胞亚群。通过检测CHB患者外周血样本,发现CHB患者分泌GM-CSF、GM-CSF^+TNF-α^+、GMCSF^+IFN-γ^+的Th细胞亚群百分率均显著高于健康对照组,差异具有统计学意义(t=2.576、4.208、2.671,P均<0.05)。结论建立的多色流式细胞因子胞内染色方案可用于明确CHB患者外周血分泌双重细胞因子的Th细胞亚群的分布模式;双重细胞因子分泌的Th细胞亚群的检测可能为临床上CHB患者免疫状态的评估提供可靠依据。Objective To evaluate the expression pattern and levels of multiple cytokines among T helper cells in the peripheral blood of patients with chronic hepatitis B （CHB） in immune clearance stages by multi-color fow cytometry analysis. Methods Total of 10 cases with CHB in immune clearance stages and 10 healthy donors in Beijing Ditan Hospital, Capital Medical University from March 2017 to April 2017 were selected. Monoclonal antibodies CD3, CD4, CD8, GM-CSF, TNF-α and IFN-γ were used to establish the six-color flow cytometry analysis panel. The peripheral blood mononuclear cell （PBMC） sample from one healthy donor was applied to adjust the optimal detection voltage, fuorescence compensation and FMO control. Compared with anti-CD3/28 mAbs, the PMA-Ionomycin was identifed as an ideal stimulant for intracellular cytokine staining. PBMCs were isolated from 10 CHB patients and cultured with PMA-Ionomycin for 5 hours. Targeting cells were stained via intracellular cytokines staining procedure. The cells were acquired by multi-color fow cytometre LSR Fortessa and were analyzed by Flowjo 10.0 cytometry analysis software. The positive propotions were compared between CHB patients and healthy donors by Graph Pad Prism7.0 software. Results The multi-color flow cytometry intracellular cytokine staining procedure was established for the functional evaluation of T cells. We frst set up the gating strategy to analyze the co-expression pattern of GM-CSF, TNF-α, IFN-γ and therefore confrmed the presence of GM-CSF＋TNF-α＋, GM-CSF＋IFN-γ＋ Th cells subsets. Simultaneously, the proportion of GM-CSF＋, GM-CSF＋TNF-α＋ and GM-CSF＋IFN-γ＋Th cells were signifcantly higher in CHB patients compared with healthy donors （t = 2.576, 4.208, 2.671; P 〈 0.05）. Conclusions The multi-color intracellular cytokine staining procedure with fow cytometry technique can be used to analyze the multiple cytokine producing pattern among Th cells subsets in CHB patients. The proportion of GM-CSF＋TNF-α＋ and GM-CSF＋IFN-γ�

关 键 词：肝炎 乙型 慢性 多色流式细胞术 细胞因子 TH细胞亚群 

分 类 号：R512.62[医药卫生—内科学]