嵌合抗原受体修饰T细胞免疫治疗食管癌的实验研究

Experimental Study of Immunotherapy of Esophageal Cancer by Chimeric Antigen Receptor-Modified T Cells

目的 观察嵌合抗原受体修饰T细胞(CAR-T)对人食管癌细胞EC109的体内外抗肿瘤作用。方法 以表皮生长因子受体Ⅲ型突变体(EGFRvⅢ)为分子靶点、EC109-EGFRvⅢ为靶细胞,将二代CAR表达框克隆入慢病毒表达载体中,制备慢病毒并转染人外周血T细胞,采用乳酸脱氢酶(LDH)活性及干扰素γ(IFN-γ)等方法检测体外抗肿瘤作用,通过NSG小鼠移植瘤模型观察体内抗肿瘤作用。结果 表达质粒pEGFRvⅢ/CAR经限制性内切酶片段分析、基因检测及NCBI/Blast同源性分析确认各功能单位核苷酸序列准确无误、基因片段连接正确,流式细胞术检测慢病毒转染后T细胞EGFRvⅢ/CAR表达率为82%。效应细胞(EGFRvⅢ/CAR-T)和靶细胞(EC109-EGFRvⅢ)混合培养,LDH细胞毒性检测结果显示靶细胞裂解死亡与效/靶比呈正相关(E∶T为5∶1、10∶1、20∶1、40∶1),IFN-γ在E∶T为5∶1时与对照组相比差异有统计学意义(P<0.01)。NSG小鼠皮下移植模型提示EGFRvⅢ/CAR-T对移植瘤有肿瘤抑制作用。结论 体外和体内实验显示CAR-T细胞对EC109-EGFRvⅢ有靶向杀伤作用,这为食管癌细胞免疫治疗提供了实验依据和研究基础。

Objective To observe the antitumor effects of chimeric antigen receptor modified T cells(CAR-T)on human esophageal carcinoma cell EC109 in vitro and in vivo.Methods Epidermal growth factor receptor variantⅢ(EGFRvⅢ)was selected as molecular target and EC109-EGFRvⅢas target cells,the second-generation CAR expression frame was cloned into lentivirus expression vector.Lentivirus was prepared and transfected into human peripheral blood T cells.The antitumor effect in vitro was detected by lactate dehydrogenase(LDH)activity and interferonγ(IFN-γ).The antitumor effect in vivo was observed by NSG mouse transplanted tumor model.Results The expression plasmid pEGFRvⅢ/CAR was analyzed by restriction endonucliase fragment analysis,gene detection and NCBI/Blast homology analysis.The nucleotide sequence of each functional unit was correct and the gene fragment connection was correct.The expression rate of EGFRvⅢ/CAR in T cells transfected with lentivirus was 82%by flow cytometry.The combination culture of effector cells(EGFRvⅢ/CAR-T)and target cells(EC109-EGFRvⅢ)showed that the lytic death of target cells was positively correlated with the efficiency/target ratio(E∶T was 5∶1,10∶1,20∶1,40∶1).The E∶T ratio of 5∶1 was significantly different from that of the control group(P<0.01).The subcutaneous transplantation model of NSG mice suggested that EGFRvⅢ/CAR-T had tumor inhibitory effect on transplanted tumors.Conclusion Experiments in vitro and in vivo showed that CAR-T cells have a targeted killing effect on EC109-EGFRvⅢ,which provides experimental basis and research basis for the immunotherapy of esophageal cancer cells.