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目的 获得一种靶向双唾液酸神经节苷脂（GD2）、并含有巨噬细胞特异性激活信号接头蛋白DNAX激活蛋白12(DAP12)胞内域的嵌合抗原受体巨噬细胞（CAR-M）。方法 设计并合成以DAP12胞内域为信号模块、靶向GD2的嵌合抗原受体（CAR）分子[Anti-GD2 CAR(DAP12)]，并将其克隆至腺病毒载体中。用该病毒感染经佛波酯（PMA）诱导分化的THP-1巨噬细胞，构建含有绿色荧光标记的Anti-GD2 CAR THP-1。通过活细胞工作站检测CAR分子的表达情况，并通过体外共培养实验，检测空载体组、分化簇3ζ链（CD3ζ）组和DAP12组对GD2高表达（T98G）及低表达（U251）胶质瘤细胞的吞噬功能。结果 CAR腺病毒高效率转染THP-1-M，获得Anti-GD2 CAR-M。与胶质瘤细胞共培养后，流式细胞术结果显示，与Empty组相比，Anti-GD2 CAR-M(CD3ζ)组表现出增强的吞噬作用（P<0.05）；与Anti-GD2 CAR-M(CD3ζ)组相比，Anti-GD2 CAR-M(DAP12)组对GD2高表达的T98G细胞吞噬能力进一步增强（P<0.05）。结论 通过构建Anti-GD2 CAR(DAP12)腺病毒并转染经PMA诱导分化的THP-1巨噬细胞，成功构建了靶向Anti-GD2 CAR-M(DAP12)，且其能通过特异性靶向吞噬GD2高表达胶质瘤细胞。
Abstract：Objective To obtain a construct chimeric antigen receptor macrophages(CAR-M) targeting disialoganglioside GD2(GD2) and harboring the intracellular domain of macrophage-specific activating signaling adaptor protein DNAX-activating protein 12(DAP12).Methods A CAR molecule targeting GD2 with the DAP12 intracellular domain as the signaling module [Anti-GD2 CAR(DAP12)] was designed, synthesized, and cloned into an adenoviral vector. This virus was used to infect PMA-differentiated THP-1 macrophages to construct green fluorescent protein(GFP)-labeled Anti-GD2 CAR THP-1 cells. CAR expression was confirmed using live-cell imaging. In vitro co-culture assays were performed to evaluate the phagocytic function of empty, Anti-GD2 CAR-M(CD3ζ), and Anti-GD2 CAR-M(DAP12) groups against glioma cells with high(T98G) and low(U251) GD2 expression.Results The CAR adenovirus efficiently transduced THP-1 macrophages, resulting in successful generation of Anti-GD2 CAR-M. Flow cytometry analysis following co-culture with glioma cells showed that compared to the empty group, the Anti-GD2 CAR-M(CD3ζ) group exhibited significantly enhanced phagocytosis(P<0. 05). Furthermore, compared to the Anti-GD2 CAR-M(CD3ζ) group, the Anti-GD2 CAR-M(DAP12) group demonstrated a further increase in phagocytic capacity specifically against GD2-high T98G cells(P<0. 05).Conclusion AntiGD2 CAR-M(DAP12) cells are successfully established by transducing PMA-differentiated THP-1-M with recombinant Anti-GD2 CAR(DAP12) adenovirus. The engineered CAR-M can specifically target phagocytose glioma cells with high GD2 expression.
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基本信息:
DOI：10.19405/j.cnki.issn1000-1492.2026.08.002
中图分类号:R730.51
引用信息:
[1]邱佳琪,朱梦娟,陈欣蕾,等.一种基于DAP12胞内域的靶向GD2 CAR THP-1细胞系的构建[J].安徽医科大学学报,2026,61(08):1342-1347.DOI:10.19405/j.cnki.issn1000-1492.2026.08.002.
基金信息:
国家自然科学基金项目（编号：82373877）~~
2026-07-09
2026-07-09
2026-07-09