目的： 研究小鼠T淋巴细胞在转染嵌合性T细胞受体基因后的体外抗肿瘤作用。方法： 应用重组DNA技术，将HER2特异性嵌合性T-细胞受体构建入逆转录病毒载体；转入包装细胞后，收集病毒上清，转染小鼠T淋巴细胞，转基因后的小鼠T淋巴细胞分别与HER2阳性（SK-OV-3）或阴性（MCF-7）的肿瘤细胞系共培养，检测其细胞因子γ干扰素释放，51Cr释放法检测CTL评价其抗肿瘤效应。结果： 所构建载体经酶切鉴定符合要求，乒乓法转染包装细胞系GP+E86，检测病毒滴度为1.2×106，Retronectin结合离心法转染经抗CD3/CD28单抗活化的小鼠T淋巴细胞，转染效率可达50%以上；转染嵌合性T细胞受体基因的T淋巴细胞与HER2阳性或阴性的肿瘤细胞系共培养后可检测到HER2特异性的细胞因子γ干扰素释放，51Cr释放法测CTL可见转染嵌合性T细胞受体基因T淋巴细胞对HER2阳性的肿瘤细胞具显著杀伤效应。结论： 转染嵌合性T细胞受体基因的小鼠T淋巴细胞在体外可通过细胞因子释放和CTL效应发挥显著的抗肿瘤作用。

Objective： To investigate the anti-tumor effects of murine T-lymphocytes harboring p185HER2 specific chimeric T-cell receptor gene.Methods: The p185HER2 specific chimeric T-cell receptor N29γ or N29ζ were introduced into retroviral vector pRET6, and the viral producer cell line was established using a Ping-Pong method. Murine spleen T-lymphocytes were transfected using an optimized protocol incorporating activation with immobilized anti-CD3/anti-CD28, followed by infection in the presence of Retronectin. Transduced murine T-lymphocytes were co-cultured with tumor cell lines overexpressing (SK-OV-3) or underexpressing (MCF-7) p185HER2 for assaying antigen specific cytokine release and CTL. Results: Endonuclease digestion showed the constructed vector was corrected. The titer of retroviral supernatant collected was 1.2×106 and the retroviral transduction efficiency reached over 50% with our optimized method. Both N29γ and N29ζ chimeric T-cell receptor transduced T-lymphocytes demonstrated HER2-specific antigen response as determined by release of interferon γ and cellular cytotoxicity assays. Conclusion: Our results suggest that murine T-lymphocytes harboring chimeric T-cell receptor gene had obvious antitumor effect in vitro through releasing cytokines and CTL effect.