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

doi:10.6040/j.issn.1673-3770.0.2017.017

山东大学耳鼻喉眼学报 ›› 2017, Vol. 31 ›› Issue (2): 49-54.doi: 10.6040/j.issn.1673-3770.0.2017.017

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

周荧¹,徐亚如²,黄骁辰²,陈渊¹,章明炯²,张大为¹,唐奇²,朱进³,陈仁杰¹

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

收稿日期:2017-01-11 出版日期:2017-04-16 发布日期:2017-04-16
通讯作者: 陈仁杰. E-mail:renjiechenent@aliyun.com
基金资助:
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)

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

1. Deparment of Otolaryngology, the Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, Jiangsu, China;2. Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University, Nanjing 210029, Jiangsu, China;3. Medical Research Institute of Nanjing Military Region, Nanjing 210002, Jiangsu, China

Received:2017-01-11 Online:2017-04-16 Published:2017-04-16

摘要/Abstract

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-MetCAR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。

关键词: 鼻咽肿瘤, c-Met, 嵌合抗原受体

Abstract: 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 scFv, 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.

Key words: Nasopharyngeal carcinoma, c-Met, Chimeric antigen receptor

中图分类号:

R739.62

周荧,徐亚如,黄骁辰,陈渊,章明炯,张大为,唐奇,朱进,陈仁杰. 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[J]. 山东大学耳鼻喉眼学报, 2017, 31(2): 49-54.

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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.

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