疫苗诱导的特异性细胞杀伤活性检测方法的建立

Development of a method for determination of vaccine-induced specific cellular killing activity

目的建立一种基于流式细胞术的评价疫苗诱导的特异性细胞杀伤活性的方法,以完善疫苗及基因治疗药物的评价方法。方法用羧基荧光素二乙酸盐琥珀酰亚胺酯(Carboxyfluorescein diacetate,succinimidyl ester,CFSE)标记淋巴细胞,利用肿瘤坏死因子(Tumor necrosis factor,TNFα)模拟杀伤淋巴细胞,用碘化丙啶(Propidium iodide,PI)染色,流式细胞仪检测,确定CFSE和PI的浓度及作用时间。利用已确知有较强细胞免疫作用的治疗性乙型肝炎疫苗免疫小鼠,分离特异性淋巴细胞并用特异性肽刺激,分离未免疫小鼠的淋巴细胞作为靶细胞,并用特异性抗原肽致敏,并从靶细胞的标记、效应细胞培养时间,效靶作用时间、效靶比几方面进行优化,确定试验方法的操作流程。结果采用CFSE/PI双标记能有效分离实验所需各组群,细胞分为CFSE+PI-、CFSE+PI+、CFSE-PI+、CFSE-PI-4个组群,可区分活细胞和凋亡细胞。CFSE标记靶细胞的时间为6 h;效应细胞培养时间为72 h;效靶作用时间为6 h;效靶比可使用100∶1和50∶1。结论建立了基于流式细胞术的评价疫苗诱导的特异性细胞杀伤活性的方法,该方法可有效和精确地评价CTL杀伤效应,完善了疫苗及基因治疗药物的评价方法。

Objective To develop a method for evaluation of vaccine-induced specific cellular killing activity based on flow cytometry（FCM）and perfect the method for assessment of vaccine and gene therapeutic drugs.Methods Lymphocytes were labeled with CFSE（carboxyfluorescein diacetate,succinimidyl ester）and treated with TNFα to mimic the cytotoxic effect of cytotoxic killer cells,then determined by FCM after propidium iodide（PI）staining,based on which the concentrations of CFSE and PI and time for treatment were optimized.Mice were immunized with therapeutic hepatitis B vaccine with known high cellular immunity,of which the specific lymphocytes were isolated from spleen and stimulated with specific peptide.The lymphocytes of unimmunized mice were isolated as target cells and sensitized with specific antigenic peptide.A procedure was developed based on the optimization of conditions including the staining of target cells,the stimulation time of effector cells,as well as the action time between target and effector cells.Results The cells were divided into CFSE＋PI-,CFSE＋PI＋,CFSE-PI＋ and CFSE-PI-groups by CFSE / PI staining,meanwhile,and survival and apoptotic cells were well distinguished.The optimal time for labeling of target cells with CFSE was 6 h,while that for stimulation of effector cells was 72 h,and the optimal action time between target and effector cells was 6 h.However,both the effector to target cell ratios of 100 ∶ 1 and 50 ∶ 1 might be adopted.Conclusion A method for evaluation of vaccine-induced specific cellular killing activity based on FCM was successfully developed,which may be used for effective and precise assessment of CTL activity and perfection of method for assessment of vaccines and therapeutic drugs.