中国肿瘤生物治疗杂志2026，Vol.33Issue(3)：233-242,10.DOI:10.3872/j.issn.1007-385x.2026.03.002
异位表达血红蛋白亚基增强CAR-T细胞在缺氧条件下的体外肿瘤细胞杀伤作用
Ectopic expression of hemoglobin subunits enhances the in vitro cytotoxicity of CAR-T cells against tumor cells under hypoxic conditions
摘要
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.关键词
实体瘤/缺氧微环境/嵌合抗原受体T细胞/血红蛋白Key words
solid tumors/hypoxia tumor microenvironment(TME)/chimeric antigen receptor T cell(CAR-T cell)/hemoglobin(Hb)分类
医药卫生引用本文复制引用
杨建勋,阎博,孙志宏,杨安钢,郑瑞,梁思辛,潘杰,李琰龙,翟晨曦,赵晓娟,王鹏举,董昊..异位表达血红蛋白亚基增强CAR-T细胞在缺氧条件下的体外肿瘤细胞杀伤作用[J].中国肿瘤生物治疗杂志,2026,33(3):233-242,10.基金项目
国家自然科学基金(82503710) （82503710）
消化系肿瘤整合防治全国重点实验室课题(CBSKL2022ZZ04,2025GTEP012) （CBSKL2022ZZ04,2025GTEP012）
陕西省重点项目(2025SYS-SYSZD-030) （2025SYS-SYSZD-030）
