摘要
鉴于嵌合抗原受体T细胞(chimeric antigen receptor T cells,CAR-T)疗法应用于肿瘤治疗的临床试验已取得很大突破,设计了程序性死亡配体-1(programmed death ligand-1,PD-L1)特异性CAR-T细胞以体外杀伤肺癌细胞。通过克隆表达PD-L1_((73-739)),并纯化PD-L1蛋白以免疫BALB/c小鼠制备获得PD-L1单克隆抗体;克隆PD-L1单克隆抗体单链可变区片段,与CD28、4-1BB、CD3-ζ链的基因体外融合构建第三代CAR基因,并克隆于慢病毒载体pCDH-CMV-EF1-copGFP上,包装成慢病毒。该慢病毒感染CD^(8+)T细胞,扩增5d,测定CAR的表达,表达率可达到22%以上。PD-L1靶向的肿瘤细胞杀伤作用分析显示,抗PD-L1CAR-T细胞有一定的体外杀伤活性。
In view of that great breakthrough has made in clinical experiment of applying Chimeric antigen receptor T cells（CAR-T）therapy,programmed death ligand-1（PD-L1）specific CAR-T cells were designed to realize killing lung cancer cells in vitro.PD-L1 monoclonal antibody was obtained by cloning and expression of PD-L1（73-739）and purifying PD-L1 protein with immune BALA/c mice;the variable region fragmentation of PD-L1 monoclonal antibody was cloned,fused with genes of CD28,4-1 BB and CD3-ζchains to construct the third generation of CARgene,and cloned onto lentiviral vector pCDH-CMVEF1-copGFP to package as lentivirus.The lentivirus was infected with CD^8＋ T cells,amplified for 5 days,and determined CAR expression（the expression rate can reach up to 22%）.The analysis of the function of PD-L1 target of killing tumor cells showed that anti-PD-L1 CAR-T cells are of certain in vitro cytotoxicity.
出处
《浙江理工大学学报（自然科学版）》
2017年第6期901-908,共8页
Journal of Zhejiang Sci-Tech University(Natural Sciences)
基金
国家高技术研究发展计划项目(2012ZX09102301-009)
浙江省自然科学基金项目(13H090015)
