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目的 构建靶向白细胞介素-13受体α(IL-13Rα)且胞内域整合可双重激活巨噬细胞与T细胞的信号接头蛋白OX40L的嵌合抗原受体巨噬细胞(CAR-M)。方法 设计并合成以OX40L为胞内信号域、靶向IL-13Rα的CAR分子(IL-13Rα-OX40LCAR)，将其定向克隆至腺病毒表达载体。采用佛波酯诱导THP-1细胞分化为巨噬细胞(THP-1-M)后，用重组腺病毒感染该细胞，构建稳定表达绿色荧光蛋白(GFP)标记的anti-IL-13Rα CAR的THP-1巨噬细胞。通过流式细胞术监测CAR分子的表达效率；以空载体组为阴性对照、CD3ζ组为阳性对照、OX40L-CD3ζ组为实验组，通过体外共培养实验，检测各组巨噬细胞对IL-13Rα高表达胶质瘤细胞(U251)及低表达胶质瘤细胞(T98G)的吞噬功能差异。结果 CAR腺病毒高效率转染THP-1-M，获得anti-IL-13Rα CAR-M。与不同IL-13Rα表达水平的胶质瘤细胞共培养后，流式细胞术结果显示，对于低表达IL-13Rα的T98G细胞，三组间差异无统计学意义；在与IL-13Rα高表达的U251细胞共培养后，与空载体组相比，CD3ζ组表现出增强的吞噬作用(P<0.05)，与CD3ζ组相比，OX40L-CD3ζ组吞噬能力进一步增强(P<0.05)。结论 通过构建IL-13Rα-OX40L-CAR腺病毒并转染经PMA诱导分化的THP-1-M，成功构建了anti-IL13Rα-OX40L-CAR-M，且其能通过特异性靶向吞噬高表达IL-13Rα的胶质瘤细胞，并表现出优于传统CD3ζ胞内域的激活。
Abstract：Objective To construct a chimeric antigen receptor macrophage(CAR-M) targeting interleukin-13 receptor α(IL-13Rα) and intracellular domain integration, which can double activate the signal adaptor protein OX40 L of macrophages and T cells.Methods The CAR molecule targeting IL-13Rα(IL-13Rα-OX40L-CAR) with OX40L as the intracellular signal domain was designed and synthesized, and it was cloned into the adenovirus expression vector. After THP-1 cells were induced to differentiate into macrophages(THP-1-M) by phorbol ester, the cells were infected with recombinant adenovirus to construct THP-1 macrophages stably expressing green fluorescent protein(GFP) labeled anti-IL-13RαCAR. The expression efficiency of CAR molecules was monitored by flow cytometry. The empty vector group was used as the negative control, the CD3ζ group was used as the positive control, and the CD3ζ-OX40L group was used as the experimental group. The phagocytic function of macrophages in each group on IL-13Rα high expression glioma cells(U251) and low expression glioma cells(T98G) was detected by in vitro co-culture experiments.Results CAR adenovirus was efficiently transfected into THP-1-M to obtain anti-IL-13RαCAR-M. After co-cultured with glioma cells with different expression levels of IL-13Rα, the results of flow cytometry showed that there was no significant difference among the three groups for T98G cells with low expression of IL-13Rα. After co-culture with U251 cells with high expression of IL-13Rα, the CD3ζ group showed enhanced phagocytosis compared with the empty vector group(P<0. 05). Compared with the CD3ζ group, the phagocytosis ability of the CD3ζ-OX40L group was further enhanced(P<0. 05).Conclusion Anti-IL13Rα-OX40L-CAR-M is successfully constructed by constructing IL-13Rα-OX40L-CAR adenovirus and transfecting PMA-induced differentiated THP-1-M. It can engulf glioma cells with high expression of IL-13Rα by specific targeting, and shows activation of intracellular domain superior to traditional CD3ζ.
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基本信息:
DOI：10.19405/j.cnki.issn1000-1492.2026.07.002
中图分类号:R392
引用信息:
[1]薛慧,薛梓萌,陈维乐,等.一种基于OX40L胞内域的靶向IL-13Rα CAR巨噬细胞的构建与验证[J].安徽医科大学学报,2026,61(07):1170-1175.DOI:10.19405/j.cnki.issn1000-1492.2026.07.002.
基金信息:
国家自然科学基金项目(编号：82373877)~~
2026-05-14
2026-05-14
2026-05-14