【摘要】：目的:1.建立活細胞染料羧基熒光素乙酰乙酸琥珀酰亞胺酯(Carboxyfluorescein diacetate,succinimidyl ester,CFDA-SE,也簡稱為CFSE)染色流式細胞術檢測淋巴細胞增殖的方法。2.應用活細胞熒光染料CFSE染色和流式細胞術檢測方法觀察不同激活劑包括結核桿菌抗原(Mtb-Ag)激活人外周血T細胞后各亞群的增殖反應動力學。3.觀察Mtb-Ag激活人外周血中γδT細胞產生白介素2(IL-2)和γ-干擾素(IFN-γ)與細胞增殖動力學的相互關系。 方法:1.常規(guī)分離PBMC,經CFSE染色后,制成濃度為1～2×10~6/ml細胞懸液。分以結核桿菌抗原(Mtb-Ag),植物血凝素(PHA),抗CD3單抗(CD3mAb)刺激,加IL-2擴增5-12d,用PE標記T細胞亞群表面分子單抗染色,最后用流式細胞儀檢測活化后T細胞各亞群的表型及其所占比例,并用ModFit軟件分析T細胞各亞群的增殖動力模型。2.用CFSE對Mtb-Ag活化培養(yǎng)4d后的外周血淋巴細胞染色,5%CO_2,37℃培養(yǎng)72小時后,上流式細胞儀檢測γδT細胞增殖情況,同時用佛波醇脂(PMA)和離子霉素(Ionomycin)繼續(xù)刺激活化,Monesin阻斷細胞內因子向細胞外的分泌,檢測細胞因子IL-2及IFN-γ的分泌情況。 結果:1.PHA和CD3mAb主要活化總T細胞,活化5天后,CD_3~+T細胞在PBMC中占到90%以上,CD_4~+T細胞和CD_8~+T細胞出現明顯的分化,CD_8~+T細胞的增殖優(yōu)先于CD_4~+T細胞。用ModFit軟件分析,CD_4~+T細胞前四代比例大于CD_8~+T細胞,第五第六代的比例則明顯低于CD_8~+T細胞。2.Mtb-Ag主要刺激γδT細胞的增殖,活化12天時,γδT細胞在PBMC的比例占到57.80%,活化14天時,γδT細胞在PBMC的比例高達80%以上,ModFit軟件分析,CD_4~+T細胞分裂集中在前四代,γδT細胞則主要集中在第六到第八代。3.PBMC經結核桿菌活化,再經佛波醇脂(PMA)和離子霉素(ionomycin)刺激,γδT細胞分泌IFN-γ的能力強于分泌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 緯 (IFN- 緯) production by human peripheral blood 緯 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，

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