缺氧外泌体对肝癌Huh7细胞增殖、迁移及侵袭的影响

Effects of hypoxic exosomes on the proliferation,migration and invasion of hepatocellular carcinoma Huh7 cells

目的探索Huh7肝癌细胞缺氧培养下分泌的外泌体对常氧Huh7肝癌细胞增殖、迁移及侵袭的影响。方法通过差速超速离心提取缺氧条件下Huh7细胞分泌的缺氧外泌体,利用透射电镜、纳米颗粒跟踪分析以及Western blot实验来鉴定缺氧外泌体。将缺氧外泌体与Huh7细胞在常氧下共培养后,分别采用CCK8、细胞划痕、Transwell小室实验检测细胞增殖、迁移及侵袭能力的变化。采用单因素方差分析和t检验进行统计学分析。结果缺氧的Huh7细胞分泌的外泌体大小在30~150nm之间,且表达有外泌体表面标志物CD9、CD63和TSG101。缺氧外泌体可显著增强常氧Huh7细胞的增殖(48h:缺氧外泌体组与对照组A值分别为2.131±0.092与1.760±0.104,t=3.740,P<0.01;72h:缺氧外泌体组与对照组A值分别为3.121±0.157与2.298±0.085,t=8.289,P<0.01)、迁移[24h:缺氧外泌体组与对照组的迁移距离分别为(0.37±0.06)cm与(0.19±0.05)cm,t=4.813,P<0.05;48h:缺氧外泌体组与对照组的迁移距离分别为(1.15±0.07)cm与(0.62±0.08)cm,t=8.874,P<0.05]及侵袭能力[缺氧外泌体组(123±18)个与对照组(44±12)个,t=6.203,P<0.01]。结论缺氧环境下肝癌Huh7细胞分泌的外泌体可以促进Huh7细胞的增殖、迁移及侵袭,提示缺氧外泌体介导的细胞间信息传递可能是肝癌缺氧微环境中肿瘤细胞恶性程度增加的关键机制之一。

Objective To explore the effects of hypoxic exosomes secreted from hepatocellular carcinoma Huh7 cells on the proliferation,migration and invasion under co-cultured normoxic condition.Methods Hypoxic exosomes secreted from Huh7 cells under hypoxic conditions were extracted by differential ultracentrifugation.Transmission electron microscopy,nanoparticles tracking analysis and western blot were used for the identification of hypoxic exosomes.Hypoxic exosomes were co-cultured with Huh7 cells under normoxic conditions.CCK8,cell scratch and transwell assay were used to detect the changes of cell proliferation,migration and invasion.Statistical analysis was performed by one-way ANOVA and t-test.Results Hypoxic exosomes secreted from Huh7 cells ranged in size from 30 to 150 nm in diameter,and expressed exosome surface markers CD9,CD63 and TSG101.Hypoxic exosomes significantly enhanced the proliferation of normoxic Huh7 cells(A value of hypoxic exosomes and control group at 48 and 72 h were 2.131±0.092 and 1.760±0.104,t=3.740,P<0.01,3.121±0.157 and 2.298±0.085,t=8.289,P<0.01).The migration distance between hypoxic exosome and control group at 48 and 72 h were(0.37±0.06 cm)and(0.19±0.05 cm),t=4.813,P<0.05,(1.15±0.07 cm)and(0.62±0.08 cm),t=8.874,P<0.05,and invasion ability[hypoxic exosomes and control group were(123±18),(44±12),t=6.203,P<0.01].Conclusion Hypoxic exosomes secreted from hepatocellular carcinoma Huh7 cells can promote cell proliferation,migration and invasion in hypoxic environment,suggesting that intercellular information transmission mediated by hypoxic exosomes may be one of the key mechanisms for the amplification of malignancy of hepatocellular carcinoma cells in hypoxic microenvironment.