活细胞染料CFSE在人γδT细胞增殖及分泌细胞因子研究中的应用
发布时间：2018-10-15 14:31
【摘要】：目的:1.建立活细胞染料羧基荧光素乙酰乙酸琥珀酰亚胺酯(Carboxyfluorescein diacetate,succinimidyl ester,CFDA-SE,也简称为CFSE)染色流式细胞术检测淋巴细胞增殖的方法。2.应用活细胞荧光染料CFSE染色和流式细胞术检测方法观察不同激活剂包括结核杆菌抗原(Mtb-Ag)激活人外周血T细胞后各亚群的增殖反应动力学。3.观察Mtb-Ag激活人外周血中γδT细胞产生白介素2(IL-2)和γ-干扰素(IFN-γ)与细胞增殖动力学的相互关系。 方法:1.常规分离PBMC,经CFSE染色后,制成浓度为1～2×10~6/ml细胞悬液。分以结核杆菌抗原(Mtb-Ag),植物血凝素(PHA),抗CD3单抗(CD3mAb)刺激,加IL-2扩增5-12d,用PE标记T细胞亚群表面分子单抗染色,最后用流式细胞仪检测活化后T细胞各亚群的表型及其所占比例,并用ModFit软件分析T细胞各亚群的增殖动力模型。2.用CFSE对Mtb-Ag活化培养4d后的外周血淋巴细胞染色,5%CO_2,37℃培养72小时后,上流式细胞仪检测γδT细胞增殖情况,同时用佛波醇脂(PMA)和离子霉素(Ionomycin)继续刺激活化,Monesin阻断细胞内因子向细胞外的分泌,检测细胞因子IL-2及IFN-γ的分泌情况。 结果:1.PHA和CD3mAb主要活化总T细胞,活化5天后,CD_3~+T细胞在PBMC中占到90%以上,CD_4~+T细胞和CD_8~+T细胞出现明显的分化,CD_8~+T细胞的增殖优先于CD_4~+T细胞。用ModFit软件分析,CD_4~+T细胞前四代比例大于CD_8~+T细胞,第五第六代的比例则明显低于CD_8~+T细胞。2.Mtb-Ag主要刺激γδT细胞的增殖,活化12天时,γδT细胞在PBMC的比例占到57.80%,活化14天时,γδT细胞在PBMC的比例高达80%以上,ModFit软件分析,CD_4~+T细胞分裂集中在前四代,γδT细胞则主要集中在第六到第八代。3.PBMC经结核杆菌活化,再经佛波醇脂(PMA)和离子霉素(ionomycin)刺激,γδT细胞分泌IFN-γ的能力强于分泌IL-2
[Abstract]:Objective: 1. To establish a method for the detection of lymphocyte proliferation by flow cytometry with live cell dye carboxyl fluorescein acetoacetate succinimide (Carboxyfluorescein diacetate,succinimidyl ester,CFDA-SE, or CFSE) staining. 2. The proliferation kinetics of T cells activated by different activators, including Mycobacterium tuberculosis antigen (Mtb-Ag), was observed by living cell fluorescent dye CFSE staining and flow cytometry. 3. To observe the relationship between Mtb-Ag activation of interleukin 2 (IL-2) and interferon 纬 (IFN- 纬) production by human peripheral blood 纬 未 T cells and cell proliferation kinetics. Methods: 1. Conventional PBMC, was stained with CFSE, and the cell suspension was obtained at the concentration of 1g 2 脳 10~6/ml. Mycobacterium tuberculosis antigen (Mtb-Ag), phytohemagglutinin (PHA), anti-CD3 monoclonal antibody (CD3mAb), IL-2 amplification for 5-12 days, and PE labeled T cell subgroup surface molecular monoclonal antibody staining were used to detect the phenotype and proportion of activated T cell subsets by flow cytometry. The dynamic model of T cell proliferation was analyzed by ModFit software. 2. The peripheral blood lymphocytes were stained with CFSE for 4 days after Mtb-Ag activation and cultured at 37 鈩，
本文编号：2272845
[Abstract]:Objective: 1. To establish a method for the detection of lymphocyte proliferation by flow cytometry with live cell dye carboxyl fluorescein acetoacetate succinimide (Carboxyfluorescein diacetate,succinimidyl ester,CFDA-SE, or CFSE) staining. 2. The proliferation kinetics of T cells activated by different activators, including Mycobacterium tuberculosis antigen (Mtb-Ag), was observed by living cell fluorescent dye CFSE staining and flow cytometry. 3. To observe the relationship between Mtb-Ag activation of interleukin 2 (IL-2) and interferon 纬 (IFN- 纬) production by human peripheral blood 纬 未 T cells and cell proliferation kinetics. Methods: 1. Conventional PBMC, was stained with CFSE, and the cell suspension was obtained at the concentration of 1g 2 脳 10~6/ml. Mycobacterium tuberculosis antigen (Mtb-Ag), phytohemagglutinin (PHA), anti-CD3 monoclonal antibody (CD3mAb), IL-2 amplification for 5-12 days, and PE labeled T cell subgroup surface molecular monoclonal antibody staining were used to detect the phenotype and proportion of activated T cell subsets by flow cytometry. The dynamic model of T cell proliferation was analyzed by ModFit software. 2. The peripheral blood lymphocytes were stained with CFSE for 4 days after Mtb-Ag activation and cultured at 37 鈩，
本文编号：2272845
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