靶向c-Met嵌合抗原受体T细胞的制备及其对鼻咽癌细胞作用的研究  

Construction of c-Met target chimeric antigen receptor T cells and evaluation the role on nasopharyngeal carcinoma cells

作　　者：周荧[1] 徐亚如 黄骁辰 陈渊[1] 章明炯 张大为[1] 唐奇[2] 朱进[3] 陈仁杰[1] 

机构地区：[1]南京医科大学第二附属医院耳鼻喉科,江苏南京210011 [2]南京医科大学卫生部抗体技术重点实验室,江苏南京210029 [3]南京军区军事医学研究所,江苏南京210002

出　　处：《山东大学耳鼻喉眼学报》2017年第2期49-54,共6页Journal of Otolaryngology and Ophthalmology of Shandong University

基　　金：江苏省科技厅临床医学科技专项计划(BL2013038);国家青年科学基金资助项目(81502673)

摘　　要：目的构建靶向c-Met的嵌合抗原受体(CAR)逆转录病毒载体,制备靶向c-Met CAR-T,观察其对c-Met阳性鼻咽癌细胞的杀伤作用。方法利用基因工程技术构建抗c-Met scFv,并将其重组到含有CD28,CD137,CD3ζ等的逆转录病毒载体中,形成c-Met CAR逆转录病毒载体,测序确认。以该病毒载体感染T淋巴细胞,通过Western blotting检测c-Met CAR在T淋巴细胞中的表达。CCK-8检测c-Met CAR-T对鼻咽癌细胞的作用;通过ELISA检测c-Met CAR-T作用后IFN-γ、IL-2的变化。结果测序结果显示c-Met CAR病毒载体序列正确;Western blotting可检测到CD3ζ的表达;CCK-8结果显示,c-Met CAR-T明显抑制c-Met阳性的鼻咽癌细胞的增殖(P<0.05);ELISA结果显示,c-Met CAR-T作用后分泌IFN-γ、IL-2明显增加(P<0.01)。结论成功制备了靶向c-Met的嵌合抗原受体逆转录病毒载体。该嵌合抗原受体能在T细胞中表达,能有效抑制c-Met阳性鼻咽癌细胞增殖,增加IFN-γ、IL-2的分泌。Objective To construct c-Met target chimeric antigen receptors（ chimeric antigen receptor,CAR） retrovirus vector and to explore its expression and effects of modified T lymphocytes on the c-Met positive nasopharyngeal carcinoma cells. Methods Genetic engineering method was used to construct the c-Met sc Fv,and then the c-Met scFv was inserted into retrovirus vector containing signaling molecules such as CD28,CD137 and CD3ζ. The build c-Met CAR retrovirus vector was confirmed by sequencing. Western blotting was used to analyze c-Met CAR expression in T lymphocyte after the virus vector infected T lymphocyte. CCK8 assay was employed to detect the cytotoxicity against the nasopharyngeal carcinoma cell lines,and changes of IFN-γ and IL-2 was analyzed by ELISA after c-Met CAR-T co-culture with nasopharyngeal carcinoma cells. Results The sequencing result showed that the c-Met CAR vector sequences were right. Western blotting could detect the expression of CD3ζ. CCK-8 assay indicated that c-Met CAR-T cells suppressed proliferation of nasopharyngeal carcinoma cells,and ELISA assay demonstrated that c-Met CAR-T cells could effectively release IFN-γ and IL-2 with the help of nasopharyngeal carcinoma cells. Conclusion c-Met CAR retrovirus vector was established successfully. The CAR could be expressed by the vector in T cells. c-Met CAR-T inhibited proliferation of c-Met positive nasopharyngeal carcinoma cells and increased the secretion of IFN-γ and IL-2.

关 键 词：嵌合抗原受体 C-MET 鼻咽肿瘤 

分 类 号：R739.62[医药卫生—肿瘤]