| 崔鑫铭,董宇曦,尚凤琴,刘秀盈,朱晶晶,刘静静,冯义超,王建勋.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[J].,2024,(1):18-24 |
| 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 |
| 投稿时间：2023-07-10 修订日期：2023-07-31 |
| DOI：10.13241/j.cnki.pmb.2024.01.004 |
| 中文关键词: B细胞成熟抗原 嵌合抗原受体 ScFv单链可变区 T细胞 |
| 英文关键词: B cell maturation antigen Chimeric antigen receptor ScFv single-chain variable region T cell |
| 基金项目:王建勋高层次人才科研启动经费项目（9011451310032） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| ABSTRACT 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 novel type of BCMA-targeting CAR-T cells by modifying the ScFv single-chain variable region of the CAR structure, which can maintain strong proliferative activity and have a stronger ability to kill tumor cells. |
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