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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 chimeric antigen receptor (CAR) retroviral vector targeting c-Met and prepare targeted c-Met CAR-T to observe its killing effect on c-Met positive nasopharyngeal carcinoma cells. Methods Anti-c-Met scFv was constructed by gene engineering and recombined into retroviral vector containing CD28, CD137 and CD3ζ to form c-Met CAR retroviral vector and confirmed by sequencing. T lymphocytes were infected with the virus vector and the expression of c-Met CAR in T lymphocytes was detected by Western blotting. The effect of c-Met CAR-T on nasopharyngeal carcinoma cells was detected by CCK-8. The changes of IFN-γ and IL-2 after c-Met CAR-T treatment were detected by ELISA. Results The sequencing results showed that the sequence of c-Met CAR was correct. The expression of CD3ζ was detected by Western blotting. The results of CCK-8 showed that c-Met CAR-T significantly inhibited the proliferation of c-Met positive NPC cells (P < 0.05). ELISA results showed that IFN-γ and IL-2 secreted by c-Met CAR-T increased significantly (P <0.01). Conclusion Chimeric antigen receptor retroviral vector targeting c-Met was successfully prepared. The chimeric antigen receptor can be expressed in T cells, which can effectively inhibit the proliferation of c-Met positive nasopharyngeal carcinoma cells and increase the secretion of IFN-γ and IL-2.