目的 外泌体（exosomes）是细胞间通信的重要媒介,研究表明卵巢癌细胞来源的外泌体可改变肿瘤微环境,但其免疫调控机制尚不明确。本研究旨在探讨卵巢癌细胞来源外泌体对树突状细胞（dendritic cells,DCs）及其介导的细胞毒性T淋巴细胞（cytotoxic T lymphocytes,CTLs）增殖与功能的影响。方法 采用超速离心法分离SKOV3细胞分泌的外泌体,利用免疫印迹法（western blotting,WB）检测外泌体标志物,动态光散射（dynamic light scattering,DLS）测定粒径分布,透射电子显微镜（transmission electron microscopy,TEM）观察形态。分离外周血单个核细胞（peripheral blood mononuclear cells,PBMCs）,分别经磷酸盐缓冲液（phosphate-buffered saline,PBS）、肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）或外泌体处理,获得未成熟、成熟及外泌体处理的DCs。将T细胞与不同组DCs共培养检测其增殖,将激活的T细胞与卵巢癌细胞进行共孵育以评估其细胞毒作用。结果 成功分离纯化SKOV3来源外泌体,TEM显示其为圆形或椭圆形双层膜囊泡,直径约50~700 nm。WB证实外泌体表达典型膜标志物及肿瘤抗原。SKOV3外泌体可显著增强DCs活化并促进初始T细胞增殖,经外泌体处理的DCs诱导的CTLs对卵巢癌细胞杀伤活性明显高于对照组。结论 卵巢癌细胞来源外泌体可促进树突状细胞活化,增强T细胞增殖及细胞毒性T淋巴细胞活性,在肿瘤免疫应答调控中具有重要作用。
Abstract
Objective Exosomes are vital mediators of intercellular communication. Studies have shown that ovarian cancer cell-derived exosomes can alter the tumor microenvironment, yet their immunomodulatory mechanisms remain unclear. This study aimed to investigate the effects of ovarian cancer cell-derived exosomes on dendritic cells (DCs) and DC-mediated cytotoxic T lymphocyte (CTLs) proliferation and function. Methods Exosomes were isolated from SKOV3 ovarian cancer cells by ultracentrifugation. Western blotting (WB) was used to detect exosomal markers, dynamic light scattering (DLS) to determine particle size distribution, and transmission electron microscopy (TEM) to observe morphology. Peripheral blood mononuclear cells (PBMCs) were treated with phosphate-buffered saline (PBS), tumor necrosis factor-α (TNF-α), or exosomes to obtain immature, mature, and exosome-treated DCs. T cells were co-cultured with different DCs groups to assess proliferation, and activated T cells were incubated with ovarian cancer cells at varying ratios to evaluate cytotoxic activity. Results SKOV3-derived exosomes were successfully isolated and purified. TEM showed round or oval bilayer vesicles with diameters of 50-700 nm. WB confirmed the presence of characteristic exosomal surface markers and tumor-associated antigens. Exosomes significantly enhanced DCs activation and promoted naïve T-cell proliferation. CTLs induced by exosome-treated DCs exhibited markedly stronger cytotoxicity against ovarian cancer cells than controls. Conclusion Ovarian cancer cell-derived exosomes promote dendritic cell activation, enhance T-cell proliferation, and strengthen cytotoxic T lymphocyte activity, suggesting an important role in regulating antitumor immune responses.
关键词
树突状细胞 /
外泌体 /
卵巢癌 /
SKOV3 /
细胞毒性 T 细胞
Key words
dendritic cells /
exosomes /
ovarian cancer /
SKOV3 /
cytotoxic tlymphocytes
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基金
湖南省卫生健康委员会科研项目“负载外泌体的树突状细胞诱导抗卵巢癌特异性T细胞的免疫效应研究” (202205015311)
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