高转移潜能细胞外泌体对肝癌细胞迁移侵袭影响及其机制  

Effect of high metastatic potential cell exosomes on the migration and invasion of hepatocellular carcinoma cells and its mechanism

作　　者：魏思佳 柳诗雅 马亮亮 孙立新 冉宇靓 孙力超 WEI Sijia;LIU Shiya;MA Liangliang;SUN Lixin;RAN Yuliang;SUN Lichao(National Cancer Center,Cancer Hospial,Chinese Academy of Medical Sciences,Peking Union Medical College,Beijing 100021,P.R.China)

机构地区：[1]国家癌症中心·国家肿瘤临床医学研究中心·中国医学科学院北京协和医学院肿瘤医院分子肿瘤学国家重点实验室,北京100021

出　　处：《中华肿瘤防治杂志》2023年第13期782-792,共11页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金面上项目(82073278);中国医学科学院医学与健康科技创新工程项目(2021-I2M-1-067);国家癌症中心中国医学科学院肿瘤医院人才激励计划(肿人发[2022]3号)。

摘　　要：目的探讨高转移潜能肝癌细胞来源的外泌体对肝癌细胞的迁移侵袭能力、干性特征及上皮-间质转化等恶性表型的影响及其可能的作用机制。方法差速离心法提取高转移潜能肝癌细胞HCCLM3的外泌体,采用透射电镜(TEM)、蛋白质印迹、纳米颗粒跟踪分析(NTA)实验对外泌体进行鉴定,免疫荧光实验检测高转移肝癌细胞外泌体可以被肝癌细胞内吞而发挥作用;分别采用无血清成球实验、Transwell实验、CCK-8实验、克隆形成实验检测高转移肝癌细胞外泌体对肝癌细胞的自我更新、迁移侵袭和增殖能力的影响;蛋白质印迹实验检测高转移肝癌细胞外泌体处理后肝癌细胞中干性相关标志物以及PI3K/AKT/mTOR信号通路蛋白表达量的变化。结果高转移潜能细胞条件培养基(CM)促进肝癌细胞的迁移侵袭和自我更新能力。在MHCC97L细胞中,迁移结果显示,高转移条件培养基组迁移数(92.33±5.03)明显高于正常培养基组(57.66±4.04)和移除外泌体条件培养基组(32.33±3.05),F=160.10,P<0.001;侵袭结果显示,高转移条件培养基组迁移数(105.33±12.58)高于正常培养基组(65.33±9.01)和移除外泌体条件培养基组(26.00±4.35),F=54.75,P<0.001。在Bel-7402细胞中,迁移结果显示,高转移条件培养基组迁移数(143.66±23.00)明显高于正常培养基组(73.33±12.58)和移除外泌体条件培养基组(38.00±6.08),F=35.94,P<0.001;侵袭结果显示,高转移条件培养基组迁移数(155.33±6.65)也显著高于正常培养基组(75.33±10.21)和移除外泌体培养基组(38.00±3.61),F=200.00,P<0.001。无血清成球结果显示,在MHCC97L细胞中,高转移条件培养基组成球个数(165.66±19.21)高于正常培养基组(113.0±14.17)和移除外泌体条件培养基组(74.33±7.76),F=29.99,P<0.001;在Bel-7402细胞中,高转移条件培养组(165.66±28.02)也显著高于正常培养基组(99.66±11.67)及移除外泌体条件培养基组(63.00±7.21),F=25.02,P<0.01。�Objective To investigate the effects of exosomes of high metastatic potential hepatocellular carcinoma cell origin on the malignant phenotype of hepatocellular carcinoma cells, including migration and invasion ability, stemness characteristics and epithelial-mesenchymal transition, and their possible mechanisms of action.Methods Exosomes of high metastatic potential hepatocellular carcinoma cells HCCLM3 were extracted by differential centrifugation and identified by transmission electron microscopy(TEM),western blotting, and nanoparticle tracking analysis(NTA) experiments.Immunofluorescence assay was used to detect that exosomes of highly metastatic hepatocellular carcinoma cells can be endocytosed by hepatocellular carcinoma cells and function;serum-free sphere-forming assay, transwell assay, CCK-8 assay and clonogenesis assay were used to detect the effects of exosomes of highly metastatic hepatocellular carcinoma cells on self-renewal, migration invasion and proliferation of hepatocellular carcinoma cells.Western blotting assays were performed to detect changes in stemness-related markers and protein expression of PI3K/AKT/mTOR signaling pathway in hepatocellular carcinoma cells after exosome treatment of highly metastatic hepatocellular carcinoma cells.Results High metastatic potential cell conditioned medium(CM) promoted the migratory invasion and self-renewal ability of hepatocellular carcinoma cells.In MHCC97L cells, the migration results showed that the migration number in the high metastatic conditioned medium group(92.33±5.03) was significantly higher than that in the normal medium group(57.66±4.04) and the migrated exosome conditioned medium group(32.33±3.05;F=160.10,P<0.001);the invasion results showed that the migration number in the high metastatic conditioned medium group(105.33±12.58) was higher than that of normal medium group(65.33±9.01) and exocytosis conditioned medium group(26.00±4.35;F=54.75,P<0.001).In Bel-7402 cells, the migration results showed that the migration number of the high

关 键 词：肝癌细胞 外泌体 干性特性 迁移侵袭 上皮-间质转化能力 

分 类 号：R735.7[医药卫生—肿瘤]