外泌体调控巨噬细胞极化对人食管鳞状细胞癌细胞增殖和转移的促进作用及其机制

doi:10.13481/j.1671-587X.20260411

摘要/Abstract

摘要：

目的探讨外泌体诱导巨噬细胞向M2型极化对人食管鳞状细胞癌（ESCC）细胞生物学行为的影响，并阐明其作用机制。方法基于癌症基因组图谱（TCGA）数据库中食管癌和正常食管组织数据，应用基因表达谱交互式分析（GEPIA）鉴定不同亚型肿瘤相关巨噬细胞（TAMs）在食管癌和正常组织中的富集情况及食管癌组织中缺氧诱导因子1α（HIF-1α）与相关分子表达的相关性，采用肿瘤免疫估计资源（TIMER）软件分析TAMs对食管癌患者生存指标的影响、单样本基因集富集分析（ssGSEA）筛选M2型TAMs极化相关基因。在基因表达综合（GEO）数据库中以“ESCC”和“外泌体”为关键词进行检索，获得GSE104926数据集，包括ESCC患者和非ESCC患者血浆外泌体基因表达谱数据。利用GEO2R分析差异基因，采用外泌体分离试剂盒提取和纯化人ESCC细胞系EC109产生的外泌体。采用透射电子显微镜（TEM）和纳米颗粒跟踪分析技术（NTA）检测外泌体形态表现和粒径，Western blotting法检测提纯的外泌体和纯化后的EC109上清液中外泌体相关蛋白分化簇（CD）9和CD63的表达情况。应用佛波酯诱导人外周血单核细胞THP-1为M0型巨噬细胞，免疫荧光实验观察外泌体被巨噬细胞内化的过程，流式细胞术检测EC109外泌体处理后的巨噬细胞和未处理的M0型巨噬细胞的表型标记物CD80和CD206表达。缺氧条件下，采用实时荧光定量PCR（RT-qPCR）法和Western blotting法检测EC109外泌体处理前后巨噬细胞中HIF-1α mRNA和蛋白表达水平。将外泌体诱导前后的巨噬细胞与EC109细胞共培养，采用细胞计数盒8（CCK-8）法检测EC109细胞增殖活性，流式细胞术检测EC109细胞凋亡率，Transwell小室实验检测EC109细胞的侵袭细胞率，划痕实验检测EC109细胞迁移能力，并计算划痕愈合率。结果 TCGA和GEO数据分析，与正常食管组织比较，食管癌组织中各亚型TAMs浸润数均明显增加（P<0.05）；M2型TAMs浸润数与食管癌患者无疾病间隔时间（DFI）有关联（P<0.05）。双糖链蛋白聚糖（BGN）为在ESCC患者血浆外泌体中表达水平升高，且与M2型TAMs极化有关联（P<0.05）。在食管癌组织中，M2型TAMs中标记物巨噬细胞清道夫受体1（MSR1）、BGN与HIF-1α表达水平存在明显正相关关系（r>0，P<0.05）。分离的EC109衍生的外泌体形态呈茶托状，大小为（127.17±0.90）nm。与提取后的EC109上清液比较，提取的EC109外泌体中CD9和CD63表达水平明显升高（P<0.05）。EC109外泌体可被巨噬细胞摄取并内化。与未处理的M0型巨噬细胞比较，EC109外泌体处理后的巨噬细胞中CD80和CD206阳性细胞数明显增多（P<0.05）；细胞中HIF-1α mRNA和蛋白表达水平均明显升高（P<0.05）。与M₀型巨噬细胞处理的EC109细胞比较，外泌体诱导的巨噬细胞与EC109细胞共培养后，EC109细胞增殖活性升高（P<0.05），细胞凋亡率升高（P<0.05），侵袭细胞率升高（P<0.05），划痕愈合率升高（P<0.05）。结论 ESCC细胞衍生的外泌体可能通过BGN激活HIF-1α调控巨噬细胞极化，从而促进ESCC增殖和侵袭，并抑制凋亡，其机制与外泌体在ESCC和TAMs之间的相互作用中发挥信号传递作用有关。

关键词: 食管肿瘤, 外泌体, 肿瘤相关巨噬细胞, 巨噬细胞极化, 缺氧诱导因子1α, 双糖链蛋白聚糖

Abstract:

Objective To discuss the effect of exosome-induced macrophage polarization to M2 type on the biological behavior of human esophageal squamous cell carcinoma （ESCC） cells， and to clarify its molecular mechanism. Methods The data of esophageal cancer and normal esophageal tissue from The Cancer Genome Atlas （TCGA） database were analyzed； Gene Expression Profiling Interactive Analysis （GEPIA） was used to identify the enrichment of different subtypes of tumor-associated macrophages （TAMs） in esophageal cancer and normal tissues and the correlation between hypoxia-inducible factor 1α （HIF-1α） and related molecule expression in esophageal cancer； Tumor Immune Estimation Resource （TIMER） software was used to analyze the impact of TAMs on the survival indicators of esophageal cancer patients； single-sample gene set enrichment analysis （ssGSEA） was used to screen genes related to M2-type TAMs polarization. The Gene Expression Omnibus （GEO） database was searched with the keywords “ESCC” and “exosome” to obtain the GSE104926 dataset， which included plasma exosome gene expression profile data from ESCC patients and non-ESCC patients. GEO2R was used to analyze differential genes， exosome isolation kit was used to extract and purify exosomes produced by human ESCC cell line EC109. Transmission electron microscope （TEM） and nanoparticle tracking analysis （NTA） were used to detect the morphology and particle size of exosomes； Western blotting method was used to detect the expression of exosome-related proteins cluster of differentation（CD）9 and CD63 in the purified exosomes and the purified EC109 supernatant. Human peripheral blood mononuclear cell line THP-1 was induced into M0 macrophages using phorbol ester； immunofluorescence staining was used to observe the internalization process of exosomes by macrophages； flow cytometry was used to detect the expression of phenotypic markers CD80 and CD206 in macrophages after treated with EC109 exosomes and untreated M0 macrophages. Under hypoxic conditions， real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods were used to detect the expression levels of HIF-1α mRNA and protein in macrophages before and after EC109 exosome treatment. The macrophages before and after exosome induction were co-cultured with EC109 cells； cell counting kit 8（CCK-8） method was used to detect the proliferation activity of the EC109 cells； flow cytometry was used to detect the apoptotic rate of the EC109 cells； Transwell chamber assay was used to detect the rate of invasion EC109 cells； scratch wound healing assay was used to detect the migration ability of the EC109 cells and the wound closure rate was calculated. Results The TCGA and GEO data analysis results showed that compared with normal esophageal tissue， the infiltration numbers of various subtypes of TAMs in esophageal cancer were significantly increased （P<0.05）； the infiltration number of M2-type TAMs was closely related to the disease-free interval（DFI） of esophageal cancer patients （P<0.05）. Biglycan （BGN） was a gene with increased expression in plasma exosomes of ESCC patients and significantly associated with M2-type TAMs polarization （P<0.05）. In esophageal cancer tissue， there were significant positive correlations between the expression levels of M2-type TAMs markers macrophage scavenger receptor 1 （MSR1）， biglycan（BGN） and HIF-1α （r>0， P<0.05）. The isolated EC109-derived exosomes exhibited a cup-shaped morphology with a size of （127.17±0.90） nm. Compared with extracted EC109 supernatant， the expression levels of CD9 and CD63 in the extracted EC109 exosomes were significantly increased （P<0.05）. The EC109 exosomes could be taken up and internalized by macrophages. Compared with untreated M₀ macrophages， the number of CD80^- and CD206⁺ cells in macrophages treated with EC109 exosomes was significantly increased （P<0.05）， and the expression levels of HIF-1α mRNA and protein in the cells were significantly increased （P<0.05）. Compared with M0 macrophage treatment group， after co-culture of exosome-induced macrophages with EC109 cells， the proliferation activity of the EC109 cells was increased （P<0.05）， the apoptotic rate was increased （P<0.05）， the rate of invasion cells was increased （P<0.05）， and the migration area was increased （P<0.05）. Conclusion The ESCC cell-derived exosomes may regulate macrophage polarization by activating HIF-1α through BGN， thereby promoting ESCC proliferation and invasion， and inhibiting apoptosis； its mechanism is related to the role of exosomes in signal transmission in the interaction between ESCC and TAMs.

Key words: Esophageal neoplasm, Exosome, Tumor-associated macrophage, Macrophage polarization, Hypoxia-inducible factor 1α, Biglycan

中图分类号:

R735

李疆芬,房新志. 外泌体调控巨噬细胞极化对人食管鳞状细胞癌细胞增殖和转移的促进作用及其机制[J]. 吉林大学学报(医学版), 2026, 52(4): 987-998.

Jiangfen LI,Xinzhi FANG. Promotion effect of exosome-regulated macrophage polarization on proliferation and metastasis of human esophageal squamous cell carcinoma cells and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2026, 52(4): 987-998.

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