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目的：探讨用人的脐带血单个核细胞体外同时扩增对抗ＥＢ病毒（ＥＢＶ）和巨细胞病毒（ＣＭＶ）的特异性细胞毒性Ｔ淋巴细胞的可行性。方法：利用人的脐带血单个核细胞（ＣＢＭＣ），通过ＥＢＶ感染转化成Ｂ成淋巴细胞细胞株（ＢＩＣＬ），再通过逆转录病毒载体，将ＣＭＶ蛋白基因ｐｐ６５导入ＢＬＣＬ，用这种细胞体外刺激同一供者脐带血的ＣＢＭＣ产生细胞毒性Ｔ细胞（ＣＴＬ），经［５１Ｃｒ］释放实验（ＣＲＡ）检测产生的ＣＴＬ的杀伤功能。结果：经免疫印迹（Ｗｅｓｔｅｒｎ Ｂｌｏｔ）检测，我们获得了同时表达 ＥＢＶ和ＣＷＶ特异性抗原的抗原递呈细胞 ＢＬＣＬｐｐ６５，免疫组化结果表明，Ｂｌｃｌｐｐ６５细胞表达ＣＭＶｐｐ６５抗原的阳性率高达９５％。ＣＲＡ结果证实，用ＢＬＣＬｐｐ６５刺激产生的ＣＴＬ同时对ＥＢＶ和ＣＭＶ都有细胞毒作用。用免疫磁珠法将ＣＤ４＋和ＣＤ８＋Ｔ细胞分离后再进行ＣＲＡ，表明特异性的细胞毒性作用主要是ＣＣ８＋ＣＴＬ介导的。结论：ＢＬＣＬｐｐ６５是很好的抗原递呈细胞，在体外能同时表达ＥＢＶ和ＣＭＶ蛋白抗原，用其刺激血清病毒抗体阴性的ＣＢＭＣ，能够扩增出同时针对ＥＢＶ和ＣＭＶ的特异性ＣＴＬ，其中ＣＤ８＋ＣＴＬ起主要作用。
Objective: To investigate the feasibility of using human cord blood mononuclear cells to simultaneously amplify specific cytotoxic T lymphocytes against Epstein-Barr virus (EBV) and cytomegalovirus (CMV) in vitro. Methods: Human cord blood mononuclear cells (CBMC) were transformed into B lymphoblastoid cell line (BICL) by EBV infection. The CMV protein gene pp65 was introduced into BLCL by retroviral vector and stimulated with the cells in vitro Cytotoxic T cells (CTLs) were generated from CBMC of the same donor cord blood, and the cytotoxic activity of CTLs was tested by [51Cr] release assay (CRA). Results: Western Blot showed that the antigen presenting cells BLCLpp65 which expressed both EBV and CWV specific antigens were obtained. The results of immunohistochemistry showed that the positive rate of CMVpp65 antigen expression in Blclpp65 cells was as high as 95%. CRA results confirmed that CTLs stimulated with BLCLpp65 have cytotoxic effects on both EBV and CMV. The CD4 + and CD8 + T cells were separated by immunomagnetic beads before they were separated for CRA, indicating that the specific cytotoxicity was mainly mediated by CC8 + CTL. CONCLUSION: BLCLpp65 is a good antigen-presenting cell that can express both EBV and CMV protein antigens in vitro. It stimulates the serum-antibody-negative CBMC to amplify specific CTLs against both EBV and CMV. Among them, CD8 + CTLs main effect.