摘要
目的利用基因工程技术构建表达嵌合T细胞受体(ch-TCR)分子的逆转录病毒载体(由抗 erbB2的单链抗体和信号传导链CD3ζ组成),感染预先刺激的小鼠T细胞,使得T细胞能以MHC非限制性的方式在体外识别并杀伤肿瘤细胞,为肿瘤的过继性免疫治疗提供新思路。方法将抗erbB2的单链抗体、CD8分子绞链区、CD3ζ链的跨膜区和胞内段依次克隆入逆转录病毒载体pCMMP中,与另外两个辅助载体pHDM．G和pMD．MLVgag．pol共同转染293T细胞,包装获得病毒颗粒,感染NIH3T3细胞检测病毒滴度。取适量病毒液感染预先刺激的小鼠CD3+T细胞,流式细胞仪检测嵌合受体在细胞表面的表达,非放射性的细胞杀伤试剂盒和ELISA检测试剂盒分别检测T细胞的抗原特异性杀伤和细胞因子分泌的功能。结果成功构建抗erbB2嵌合T细胞受体基因的逆转录病毒载体,检测病毒滴度为(2．1±1．1)×108IP／ml。体外感染T淋巴细胞的效率为(45±5)％。体外能杀伤erbB2+的肿瘤细胞SK-BR-3、D2F2／E2,而对erbB2-的肿瘤细胞 MCF-7、D2F2无杀伤功能,并能分泌Th1型细胞因子GM-CSF和IFN-γ。结论表达抗erbB2嵌合T细胞受体的T淋巴细胞体外具有MHC非限制性的、erbB2抗原特异性的杀伤肿瘤细胞和分泌 Th1型细胞因子的功能,为下一步的体内实验打下基础。
Objective To construct a retroviral vector of the chimeric TCR, which comprises scFv-anti-erbB2, CD8 hinge region and CD3ζ by gene engineering technology. After infection with preactivated mouse T lymphocytes, T cells could recognize and eliminate tumor ceils in a non-major-histocompatibility-complex （MHC） restricted manner, thus providing a promising tool for tumor adoptive immunotherapy. Methods ScFv- anti-erbB2, CD8 hinge region and CD3ζ transmembrane and cytoplasmic domains were cloned into pCMMP, accompanied by aiding plasmids pHDM. G and pMD. MLVgag. pol were co-transfected into packaging cell line 293T. Viral titer was estimated by transduction of NIH3T3 cells. The preactivated mouse CD3^＋ T ceils were infected by vrials, and the cytotoxicity capacity and cytokine production of transduced CD3^＋ T cells determined by non-radioactivation cytotoxicity assay and ELISA. Results A retroviral vector of anti-erbB2-ζ chimeric TCR was constructed and expressed on mouse T ceils. Flow cytometry showed the infectious rate was （ 45 ± 5 ） % . These gene-modified T cells were able to lyse erbB2 ＋ targets and secrete high levels of IFN-γ and GM-CSF following Ag stimulation in a non-MHC restricted manner in vitro. Conclusion T lymphocytes expressing anti-erbB2 chimeric T cell receptors could kill tumor cells and secrete Th1 type cytokines in a non-MHC restricted manner and erbB2 specific manner in vitro, which lays a foundation for further experiments in vivo.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2006年第5期609-611,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目(30200331)
