论文部分内容阅读
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 establish a method for evaluating the specific cytotoxic activity induced by the vaccine based on the flow cytometry and to improve the evaluation method of the vaccine and gene therapy drugs. Methods Lymphocytes were labeled with carboxyfluorescein diacetate succinate (succinimidyl ester, CFSE). Lymphocytes were stimulated with tumor necrosis factor (TNFα) and propidium iodide, PI) staining, flow cytometry to determine the CFSE and PI concentration and duration of action. Mice were immunized with a therapeutic hepatitis B vaccine that was already proven to have a strong cellular immune response, specific lymphocytes were isolated and stimulated with specific peptides, lymphocytes of naive mice were isolated as target cells and induced with specific antigenic peptides Sensitive, and from the target cell markers, effector cell culture time, the target effect of time, target efficiency ratio optimization of several aspects to determine the test method of operation. Results CFSE / PI double labeling could effectively separate all the groups needed for the experiment. The cells were divided into four groups: CFSE + PI-, CFSE + PI +, CFSE-PI +, and CFSE-PI-4 and could distinguish between living and apoptotic cells . CFSE labeled target cells for 6 h; effector cell culture time for 72 h; target effect time of 6 h; effective target ratio can be used 100: 1 and 50: 1. Conclusion The method of evaluating the specific cytotoxic activity induced by vaccine by flow cytometry was established. This method can effectively and accurately evaluate the killing effect of CTL and improve the evaluation method of vaccine and gene therapy.