检测Epstein-Barr病毒特异性细胞毒性T淋巴细胞方法的建立及其初步研究

Establishment and primary study of an assay for the detection of EB virus specific cytotoxic T lymphocytes

目的建立一种非放射性、简便易行的可检测特异性细胞毒性Ｔ淋巴细胞的方法，并且初步应用于Ｅｐｓｔｅｉｎ－Ｂａｒ病毒的细胞免疫应答。方法用重组的ＥＢＶ－ＬＭＰ１痘苗病毒、ＴＫ＋痘苗病毒和杆状病毒系统表达的ＥＢＶ－ＬＭＰ１蛋白分别免疫Ｂａｌｂ／Ｃ小鼠，用Ｐ８１５细胞和乳酸脱氢酶法检测ＥＢ病毒特异性细胞毒性Ｔ细胞的杀伤效应。结果重组ＥＢＶ－ＬＭＰＩ痘苗病毒免疫组原发ＣＴＬ水平和体外诱生的二次ＣＴＬ水平均高于ＴＫ＋痘苗病毒免疫组和正常组；杆状病毒系统表达的ＥＢＶ－ＬＭＰ１蛋白免疫组的ＣＴＬ水平也明显高于正常鼠。结论本法可以较好的反映ＥＢ病毒特异性细胞毒性Ｔ细胞的水平，而且再一次说明ＬＭＰ１基因能够诱发特异性的细胞免疫。

In our study,a non-radioactive cytotoxicity assay by using a Balb/C (H-2 d)mice, P815(H-2 d)cells and lactate dehydrogenase (LDH)systerm was established, in order to detect EB virus specific cytotoxic T lymphocytes(CTLs). In this study,we detected both the changes of primary CTLs and secondary stimulated CTLs. Spleen CTL activity assay showed that the levels of primary and secondary CTLs in recombinant EBV-LMP1 vaccinia virus group were more higher than that in control group, at the same time, bpvious enhancement of EBV-LMP1 specific CTL activity was observed in experimental group immunized by Baculovirus system expressing EBV-LMP1 protein when compared with the normal mice group. This work proved that the LDH assay can be used to detect cytotoxic T lymphocytes.