靶向人源BCMA的嵌合抗原受体T细胞构建及体外抗肿瘤活性研究  

Construction of Chimeric Antigen Receptor T Cells Targeting Human BCMA and its Antitumor Activity in Vitro

作　　者：崔鑫铭 董宇曦 尚凤琴 刘秀盈 朱晶晶 刘静静 冯义超 王建勋 CUI Xin-ming;DONG Yu-xi;SHANG Feng-qin;LIU Xiu-ying;ZHU Jing-jing;LIU Jing-jing;FENG Yi-chao;WANG Jian-xun(School of Life Sciences,Beijing University of Chinese Medicine,Beijing,102400,China;Research Institute of Shenzhen,Beijing University of Chinese Medicine,Shenzhen,Guangdong,518118,China)

机构地区：[1]北京中医药大学生命科学学院,北京102400 [2]深圳北京中医药大学研究院,广东深圳518118

出　　处：《现代生物医学进展》2024年第1期18-24,共7页Progress in Modern Biomedicine

基　　金：王建勋高层次人才科研启动经费项目(9011451310032)。

摘　　要：目的:通过对CAR结构的ScFv单链可变区进行改造,构建并筛选具有更强杀伤肿瘤细胞功能的新型靶向人源B细胞成熟抗原(BCMA)的嵌合抗原受体(CAR)-T细胞。方法:构建靶向人源BCMA的CAR分子,用逆转录病毒载体包装成功后转导健康志愿者的T细胞,制备Anti-BCMA-CAR-T细胞。将Anti-BCMA-CAR-T细胞作为观察组,普通T细胞作为对照组,将其与RPMI-8226细胞共培养,采用CFSE染色的T细胞增殖实验观察两组体外增殖能力。采用荧光素酶化学发光实验检测两组细胞在不同效靶比(1:8、1:4、1:2、1:1、2:1、4:1)对RPMI-8226细胞的杀伤效率,采用流式细胞术检测两组细胞在不同效靶比(1:4、1:2、1:1、2:1、4:1)对RPMI-8226细胞的杀伤效率。结果:CFSE检测结果显示,与对照组比较,观察组FITC信号明显左移,表明T细胞增殖能力越强。流式细胞术检测结果显示,相同效靶比时,观察组对RPMI-8226细胞的杀伤效率均高于对照组(P均<0.05);荧光素酶化学发光实验结果显示,相同效靶比时,观察组对RPMI-8226细胞的杀伤效率均高于对照组(P均<0.05)。在效靶比为4:1时,CAR170-T(未经改造的传统的ScFv)细胞和CAR174-T(经改造的ScFv)细胞的杀伤效率分别达到了88.5±0.3%和98.5±0.7%。结论:通过对CAR结构的ScFv单链可变区进行改造后成功构建出的新型靶向BCMA的CAR-T细胞,它能保持较强的增殖活性且具有更强的杀伤肿瘤细胞的能力。Objective:To construct and screen the new type of chimeric antigen receptor CAR-T cell targeting human-derived B cell maturation antigen(BCMA),By modifying the ScFv single-chain variable region of the CAR structure,a novel chimeric antigen receptor(CAR)-T cell targeting human B-cell maturation antigen(BCMA)with a stronger ability to kill tumor cells was constructed and screened.Methods:We constructed a CAR molecule targeting BCMA human ScFv,and after successful packaging with retroviral vector,transducing T cells from healthy volunteers to prepare Anti-BCMA-CAR-T cells.Anti-BCMA-CAR-T cells were used as the observation group and normal T cells were used as the control group,they were co-cultured with RPMI-8226 cells,and the proliferation ability of the two groups in vitro was observed by the T cell proliferation experiment stained with CFSE.The killing efficiency of two groups of cells on RPMI-8226 cells at different effect-to-target ratios(1:8,1:4,1:2,1:1,2:1,4:1)was detected by luciferase chemiluminescence assay.Flow cytometry was used to detect the killing efficiency of the two groups of cells on RPMI-8226 cells at different effect-to-target ratios(1:4,1:2,1:1,2:1,4:1).Results:CFSE detection results showed that compared with the control group,the FITC signal in the observation group shifted significantly to the left,indicating that the T cell proliferation ability was stronger.The results of flow cytometry showed that at the same effect target ratio,the killing efficiency of the observation group on RPMI-8226 cells was higher than that of the control group(both P<0.05);the results of luciferase chemiluminescence experiment showed that the same effect target ratio,the killing efficiency of RPMI-8226 cells in the observation group was higher than that in the control group(P<0.05).When the effect-to-target ratio was 4:1,the killing efficiencies of CAR170-T(unmodified conventional ScFv)cells and CAR174-T(modified ScFv)cells reached 88.5±0.3%and 98.5±0.7%,respectively.Conclusion:We successfully constructed a nove

关 键 词：B细胞成熟抗原 嵌合抗原受体 ScFv单链可变区 T细胞 

分 类 号：R-33[医药卫生] R730.5