CFSE检测马传染性贫血病毒疫苗免疫马T细胞增殖方法的建立

Development of a CFSE-based flow cytometry for evaluating EIAV-stimulated proliferation of T lymphocytes

目的:以流式细胞术(FCM)检测马传染性贫血病毒(EIAV)抗原特异性的T淋巴细胞增殖。方法:将新鲜分离的马外周血单个核细胞(PBMC)进行CFSE染色,经纯化病毒体外刺激并培养5d后,进行T淋巴细胞亚型标记,检测特异性增殖的CD4+和CD8+细胞比例。结果:对马PBMC的染色浓度、特异性刺激物的种类及反应时间等条件进行了优化。可对马外周血抗原特异性T淋巴细胞不同亚型的增殖水平进行定量评价。结论:FCM检测方法为研究马传染性贫血病毒疫苗免疫保护机制提供了一个有效的手段;该法也可以为其他病毒的免疫学研究提供借鉴。

AIM： To develop a flow cytometry using （5-carboxyfluorescein diacetate succinmidyl ester, CFSE） to detect the proliferation of specific T lymphocytes from equine infectious anemia virus （EIAV）. METHODS： Peripheral blood mononuclear cells （PBMC） were isolated, stained with CFSE and incubated with EIAV for 5 days. After interacted with either CD4^＋ or CD8^＋ antibody, the cells were detected for proliferated population, which contained serially 2-fold reduced CFSE in CD4^＋ and CD8^＋ T lymphocytes. RESULTS： The concentration of CFSE, and the type, concentration and reaction time of EIAV-specific antigens were tested and optimized to improve the sensitivity and specificity of this flow cytometry-based assay. PBMCs isolated from the horses infected with either virulent or vaccine strains of EIAV and health controls were analyzed to evaluae this technique. The proliferation rates of CD4^＋ and CD8^＋ T lymphocytes were observed in teh cells from differently treated horses. CONCLUSION： The CFSE-based flow cytometry is a reliable tool to analyze antigen-specific proliferation of lymphocytes, which plays an important role in evaluating the immune responses induced by EIAV and other viruses.