[摘 要] 目的：探讨异位表达血红蛋白亚基（HBA/HBB）对缺氧条件下嵌合抗原受体T细胞（CAR-T细胞）功能障碍的改善作 用及其对肿瘤细胞的杀伤效应。方法：全基因合成技术合成靶向HER2的CAR序列，构建共表达HBA或HBB的CAR慢病毒载 体，包装慢病毒后感染人原代T淋巴细胞，制备异位表达HBA/HBB的CAR-T细胞，命名为HBA CAR-T和HBB CAR-T。采用缺 氧探针检测小鼠实体瘤缺氧状态。通过流式细胞术检测瘤内CAR-T细胞占比、异位表达血红蛋白亚基的CAR-T细胞阳性率及 CAR-T细胞的活性氧、凋亡水平。WB法检测HBA CAR-T和HBB CAR-T内相关血红蛋白亚基表达情况，采用细胞计数板计数 检测细胞增殖水平，通过萤光素酶报告基因法检测 CAR-T 细胞对肿瘤细胞的杀伤能力 ，qPCR 检测CAR-T细胞中缺氧诱导 因子-1α（HIF-1α）表达水平，利用MitoXpress Intra试剂盒检测CAR-T细胞内氧气含量。结果：不同细胞构建的实体瘤模型均存 在明显缺氧情况，且CAR-T细胞浸润水平与缺氧程度呈显著负相关（P < 0.000 1）。HBA CAR-T与HBB CAR-T构建成功（阳性 率 > 60%），相应血红蛋白亚基可稳定表达。缺氧环境下HBA CAR-T和HBB CAR-T的ROS水平、凋亡水平显著下降，增殖、对肿 瘤细胞的体外杀伤能力显著强于传统CAR-T细胞（均P < 0.05）。HBA CAR-T与HBB CAR-T内HIF-1α表达降低（均P < 0.001）， 且缺氧程度显著降低（均P < 0.001）。结论：异位表达血红蛋白亚基可改善缺氧条件下CAR-T细胞功能障碍并增强其对肿瘤细 胞的体外杀伤作用。

[Abstract] Objective: To investigate whether ectopic expression of hemoglobin subunits (HBA/HBB) improves chimeric antigen receptor T cell (CAR-T cell) function and enhances cytotoxicity against tumor cells under hypoxic conditions. Methods: The CAR sequence targeting HER2 was synthesized by full gene synthesis technology, and the CAR lentiviral vectors co-expressing HBA or HBB were constructed. After packaging the lentivirus, human primary T lymphocytes were infected to prepare HBA CAR-T and HBB CAR-T. Hypoxia in mouse solid tumors was detected by hypoxia probes. The proportion of CAR-T cells in the tumor, the percentage of CAR-T cells expressing hemoglobin, and the levels of reactive oxygen species and apoptosis of CAR-T cells under different conditions were detected by flow cytometry. The expression of related hemoglobin subunits in CAR-T cells was detected by WB assay. The proliferation level of cells was detected by hemocytometer, the cytotoxicity of CAR-T cells against tumor cells was detected by luciferase reporter assay, the expression level of HIF-1α in CAR-T cells was detected by qPCR, the oxygen content in T cells was detected by MitoXpress Intra kit. Results: The solid tumor models constructed from different cell lines all exhibited significant hypoxia, and the infiltration level of CAR-T cells was significantly negatively correlated with the degree of hypoxia (P < 0.000 1). HBA CAR-T and HBB CAR-T were successfully constructed (positive rate > 60%), and the corresponding hemoglobin subunits were stably expressed. Under hypoxic conditions, the ROS level and apoptosis level of HBA CAR-T and HBB CAR-T significantly decreased, and their proliferation and cytotoxicity against tumor cells were significantly stronger than those of conventional CAR-T cells (all P < 0.05). HBA CAR-T and HBB CAR-T showed decreased HIF-1α expression (all P < 0.001), and their level of hypoxia significantly decreased (all P < 0.001). Conclusion: The ectopic expression of hemoglobin can reverse the functional impairment of CAR-T cells under hypoxic conditions and enhance their cytotoxicity against tumor cells in vitro.

[基金项目] 国家自然科学基金（82503710）；消化系肿瘤整合防治全国重点实验室课题（CBSKL2022ZZ04, 2025GTEP012）； 陕西省重点项目（2025SYS-SYSZD-030）