流行性出血热病毒感染鼠的细胞毒T细胞功能中性红比色分析法

NEUTRAL RED COLORIMETRIC ASSAY FOR FUNCTIONAL ESTIMATION OF CYTOTOXIC T LYMPHOCYTES FROM MICE INFECTED WITH EPIDEMIC HEMORRHAGIC FEVER VIRUS

建立中性红比色分析法用于测定流行性出血热(EHF)细胞毒T细胞(CTL)功能,并对EHF地鼠肾细胞(GHKC)灭活疫苗免疫动物的CTL活性进行了初步观察。中性红比色法是用EHF病毒(EHFV)免疫主要组织相容性抗原为H-2~d的纯系Balb/C小鼠,取其脾细胞在体外用感染EHFV的同系鼠腹腔巨噬细胞刺激后作为效应细胞,以EHFV感染的同系鼠腹腔巨噬细胞经中性红染色后作为靶细胞,最后用酶联免疫检测仪测定未被杀伤的靶细胞的中性红量来分析CTL活性。结果表明,此法灵敏度高,特异性强,因不使用同位素,比较安全、经济。EHFV活毒免疫动物的CTL活性随着“效应细胞/靶细胞”(E:T)比值的增加而增加。野鼠型EHFVA9株及JR株免疫动物的CTL间表现明显的交叉免疫反应,提示同型毒株结构蛋白上存在有CTL识别的共同位点。家鼠型EHFV L99株与野鼠型JR株免疫动物的CTL间也有较低的交叉杀伤作用。以EHF-金黄色地鼠肾细胞制备的灭活疫苗免疫的动物表现有较低的类似CTL活性。

A colorimetric assay with neutral red ( NRCMA ) was established for the determination of the function of cytotoxic T lymphocytes(CTL)from epidemic hemorrhagic fever viruses ( EHFV )-infected mice and the study of CTL cytotoxic function of spleen cells of Balb/C mice immunized with inactivated golden hamster kidney cell ( GHKC ) vaccine against EHFV.Spleen cells of the immunized Balb/C mice ( H-2d) were isolated and restimulated with EHFV-infected syngeneic macrophages, and used as effector cells. Syngeneic peritoneal macrophages from normal Balb/C mice infected with EHFV, were used as the target cells. The CTL cytotoxic activity was estimated by measuring the quantity of neutral red in unkilled target cells with an ELISA detector. The method appears to be specific, sensitive, technically simple, safe and economical. It was found that the degree of CTL cytotoxicity was related directly with the ratio of effector : target cells ( E: T Ratio ) . Cross-reactive immune CTL cytotoxicity was demonstrated between different strains of the Apodemus-type EHFV, JR strain and A9 strain, suggesting that they shared commen epi topes. Lower crossreactive immunity was found between the rat-type EHFV. L99 strain and the Apodemus-type EHFV, JR strain. Similar but lower cytotoxicity of immune CTLs was demonstrated in Balb/C mice immunized with the inactivated GHKC vaccine against EHF.