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OA
Ectopic expression of hemoglobin subunits enhances the in vitro cytotoxicity of CAR-T cells against tumor cells under hypoxic conditions
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.
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.
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
延安大学 生命科学学院,陕西 延安 716000||空军军医大学 基础医学院 生物化学与分子生物学教研室,陕西 西安 710032空军军医大学 基础医学院 生物化学与分子生物学教研室,陕西 西安 710032延安大学 生命科学学院,陕西 延安 716000空军军医大学 基础医学院 免疫学教研室,陕西 西安 710032空军军医大学 基础医学院 免疫学教研室,陕西 西安 710032空军军医大学 基础医学院 免疫学教研室,陕西 西安 710032空军军医大学 西京医院妇产科,陕西 西安 710032新乡医学院 医学技术学院,河南 新乡 453003新乡医学院 医学技术学院,河南 新乡 453003空军军医大学 基础医学院 生物化学与分子生物学教研室,陕西 西安 710032空军军医大学 基础医学院 免疫学教研室,陕西 西安 710032空军军医大学 西京医院妇产科,陕西 西安 710032
医药卫生
实体瘤缺氧微环境嵌合抗原受体T细胞血红蛋白
solid tumorshypoxia tumor microenvironment(TME)chimeric antigen receptor T cell(CAR-T cell)hemoglobin(Hb)
《中国肿瘤生物治疗杂志》 2026 (3)
233-242,10
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)
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