胶质瘤内肿瘤相关巨噬细胞的缺氧转录表型及细胞超微结构改变  

Hypoxic transcriptional phenotype and cellular ultrastructural changes of tumor-associated macrophages in gliomas

作　　者：范海珍 王丽霞 程玥 王路静 阮潜瑛 吉佳乐 王梦茹 秦榛 张艺 何志承 平轶芳 时雨 FAN Haizhen;WANG Lixia;CHENG Yue;WANG Lujing;RUAN Qianying;JI Jiale;WANG Mengru;QIN Zhen;ZHANG Yi;HE Zhicheng;PING Yifang;SHI Yu(School of Medicine,Chongqing University,Chongqing;Institute of Pathology,Yu-Yue Pathology Scientific Research Center,Chongqing,China;Jinfeng Laboratory,Yu-Yue Pathology Scientific Research Center,Chongqing,China;Department of Blood Transfusion,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing)

机构地区：[1]重庆大学医学院,重庆 [2]陆军军医大学(第三军医大学)第一附属医院病理学研究所,重庆 [3]金凤实验室,渝粤病理科学研究中心 [4]陆军军医大学(第三军医大学)第一附属医院输血科,重庆

出　　处：《陆军军医大学学报》2025年第9期904-911,共8页Journal of Army Medical University

基　　金：国家重点研发计划青年科学家项目(2021YFA1103000);重庆市自然科学基金杰出青年基金项目(CSTB2023NSCQ-JQX0009);重庆市卫生健康委员会国家级杰出青年后备人才培养专项(HBRC202408)。

摘　　要：目的探讨缺氧对胶质瘤内肿瘤相关巨噬细胞(tumor-associated macrophage,TAM)转录表型及细胞超微结构的影响。方法从陆军军医大学第一附属医院血库的健康人外周血样本中分离CD14阳性单核细胞(peripheral blood mononuclear cells,PBMC),并在人胶质瘤细胞上清共培养的条件下使其分化为TAM。使用不同浓度(50~400μmol/L)的氯化钴(cobalt chloride hexahydrate,CoCl_(2))或缺氧条件(1%、5%、10%O_(2))处理48 h后诱导为TAM缺氧细胞模型,同时在相同时间下采用21%O_(2)处理为TAM常氧细胞模型。使用RT-q PCR和转录组测序技术,检测常氧组和缺氧组TAM的转录表型改变。通过基因集富集分析(gene set enrichment analysis,GSEA)比较常氧组及缺氧组TAM中血管生成、糖酵解等缺氧响应相关通路的差异。利用透射电子显微镜(transmission electron microscope,TEM)或免疫荧光染色检测常氧组TAM和缺氧组TAM(1%O_(2))的细胞骨架、内质网与线粒体的超微结构改变。结果与常氧组TAM相比,缺氧组TAM内参与氧气运输、糖酵解代谢、促血管生成相关的缺氧响应标志物的转录表达增加,且与缺氧程度相关(P<0.05)。GSEA结果显示,与常氧组TAM相比,缺氧组TAM的缺氧、血管生成调控、糖酵解与糖异生、饥饿应激通路上调(P<0.05);而先天免疫调控、巨噬细胞活化、细胞骨架与蛋白成熟通路显著下调(P<0.05)。TEM及免疫荧光染色结果提示,缺氧组TAM细胞骨架、粗面内质网、线粒体的超微结构明显改变,包括:细胞骨架排列紊乱和断裂;粗面内质网变短,表面附着核糖体减少;线粒体水肿且内嵴破环,与粗面内质网的接触减少。结论CoCl_(2)以及缺氧处理诱导TAM呈现缺氧转录表型,可能与细胞骨架、内质网和线粒体的超微结构改变有关。Objective To investigate the effects of hypoxia on the transcriptional phenotype and ultrastructure of tumor-associated macrophages (TAMs) in glioma.Methods CD14^(+) monocytes were isolated from healthy human peripheral blood samples collected from the Blood Bank of the First Affiliated Hospital of Army Medical University,and the cells were induced to differentiate into TAMs through co-culture with glioma cell-conditioned medium.Hypoxic TAM models were established using varying concentrations of cobalt chloride hexahydrate (CoCl_(2),50~400μmol/L) or hypoxic conditions (1%,5%,10%O_(2)) for 48 h,while normoxic TAM models (21%O_(2)) served as controls.RT-qPCR and transcriptome sequencing were employed to analyze transcriptional changes in TAMs under normoxic and hypoxic conditions.Gene set enrichment analysis (GSEA) was applied to compare the differences in angiogenesis,glycolysis and other hypoxiaresponsive pathways between the 2 conditions.Transmission electron microscopy (TEM) or immunofluorescence staining was conducted to assess the ultrastructural alterations in cytoskeleton,endoplasmic reticulum (ER),and mitochondria in normoxic and hypoxic TAMs (1%O_(2)).Results Hypoxic TAMs exhibited up-regulated transcription of hypoxia-responsive markers (oxygen transport,glycolysis,pro-angiogenesis),with the effects correlating with hypoxia severity(P<0.05).GSEA revealed significant up-regulation of hypoxia,angiogenesis regulation,glycolysis and gluconeogenesis,and starvation stress pathways,alongside down-regulation of innate immunity,macrophage activation,cytoskeleton,and protein maturation pathways in hypoxic TAMs (P<0.05).TEM and immunofluorescence staining demonstrated obvious ultrastructure changes,including disrupted cytoskeletal organization,shortened rough ER with reduced ribosomes,mitochondrial swelling with cristae damage,and diminished ER-mitochondria contacts in hypoxic TAMs.Conclusion CoCl_(2) and hypoxia induce a hypoxic transcriptional phenotype in TAMs,which may potentially associated with ultrastructur

关 键 词：胶质瘤 缺氧 肿瘤相关巨噬细胞 超微结构 

分 类 号：R394.3[医药卫生—医学遗传学] R730.23[医药卫生—基础医学] R730.264