表达嵌合性T细胞受体的肿瘤浸润淋巴细胞对KDR表达阳性细胞的选择性杀伤作用

The specific cytotoxic effect of tumor infiltrating lymphocytes transfected with chimeric T cell receptor on cells which express KDR

目的 研究表达嵌合性T细胞受体VEGF12 1 hinger FcRγ的肿瘤浸润淋巴细胞 (TIL)对血管内皮生长因子 (VEGF)受体KDR(kinase domaininsertcontainingreceptor)表达阳性细胞的选择性杀伤作用。方法 应用分子克隆技术重组VEGF12 1 hinger FcRγ(Vhγ) ,构建重组逆转录病毒质粒pMSCVneo Vhγ ,经PT6 7包装后筛选高滴度的病毒 ,体外感染分离于肝癌组织的TIL ,获得MSCVneo Vhγ TIL。以空病毒感染TIL获得的MSCVneo TIL作为对照。以MTT比色法分析不同的TIL细胞对 4种靶细胞 (不表达VEGF受体KDR的成纤维细胞系NIH3T3和肝癌细胞系HepG2 ,以及表达VEGF受体KDR的人脐静脉血管内皮细胞系ECV30 4和恶性黑色素瘤细胞系A375 )的杀伤活性。结果 未转染的TIL和MSCVneo TIL对NIH3T3和ECV30 4无明显的杀伤作用 ,对HepG2和A375有一定杀伤作用 ,对4种靶细胞杀伤活性的差异无显著性 ;MSCVneo Vhγ TIL对NIH3T3无明显杀伤活性 ,对ECV30 4有明显杀伤活性 ,对HepG2的杀伤与TIL、MSCVneo TIL相比差异无显著性 ,而对A375的杀伤明显增强。结论 嵌合性T细胞受体Vhγ经逆转录病毒载体介导 ,可在TIL细胞表面稳定表达 ,并获得选择性识别和溶解KDR阳性血管内皮细胞和肿瘤细胞的新效应功能。

Objective To investigate the specific cytotoxity of tumor infiltrating lymphocytes(TIL) transfected with chimeric T cell receptor (CTCR) on cells which express KDR. Methods A recombinant retroviral plasmid (pMSCVneo-Vhγ) was constructed by cloning VEGF121-hinger-FcRγ(Vhγ) into retroviral vector pMSCVneo.After packaging by PT67,the virus with high titer was used to infect TIL isolated from liver cancer tissues, and then MSCVneo- Vhγ- TIL was generated.TIL infected with MSCVneo was used as a control. The cytotoxicty of the transgenic TIL on NIH3T3 and HepG2 expressing no KDR and on ECV304 and A375 expressing KDR was detected with MTT colorimetric assay. Results The sequences of VEGF121 and hinger-FcRγ were different from those reported, but the deduced amino sequences were identical to the reported ones.The cytotoxity of TIL infected with MSCVneo on target cell was similar to that of the control TIL;both only had mild cytotoxity on cancer cell line. No significant cytotoxity was found in TIL infected with MSCVneo-cTCR on NIH3T3, but its cytotoxity on ECV304 was significant.The cytotoxity on HepG2 was similar to that of MSCVneo-TIL and uninfected TIL, but cytotoxity on A375 was significantly higher. Conclusion Chimeric T cell receptor permanently grafts TIL cell with predefined new specificity. TIL expressing Vhγ can selectively recognize and kill vascular endothelial cell and tumor cells which express VEGF receptor KDR.