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目的 观察血管内皮细胞培养上清对肝癌细胞Hep3B增殖及侵袭转移能力的影响,探讨肿瘤微环境中内皮细胞在肝癌发生、发展中的作用.方法 以人脐静脉内皮细胞(HUVEC)培养上清为条件培养液(CM),以内皮细胞培养液(EBM)为对照组,分别对Hep3B细胞进行干预.观察其对肝癌细胞Hep3B增殖、迁移和侵袭能力的影响,并检测相关信号通路分子.结果 与对照细胞比较,细胞计数试剂盒-8(CCK-8)实验结果显示CM促进肝癌细胞的增殖(P＜0.01);细胞划痕实验和迁移实验也分别显示CM明显促进肝癌细胞迁移能力[(33.65±1.22)％比(57.85±2.20)％,P ＜0.01;(51.2±3.1)个比(28.4±4.6)个,P＜0.01];侵袭实验显示CM明显诱导肝癌细胞侵袭能力[(16.2±2.3)个比(5.4±1.5)个];同时CM干预的肝癌细胞侵袭转移相关基因,如基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、骨桥蛋白(OPN)和簇分化抗原44(CD44)表达及丝氨酸/酪氨酸激酶(Akt)和细胞外信号调节激酶(ERK)蛋白磷酸化水平均被明显上调.结论 血管内皮细胞培养上清体外促进肝癌细胞增殖、迁移和侵袭转移,提示血管内皮细胞可通过旁分泌机制参与肝癌侵袭转移过程.

Objective To investigate the effects of vascular endothelial cells on the proliferation and invasion of hepatocellular carcinoma cells,and better understand the role of endothelial cells in the development of hepatocellular carcinoma (HCC).Methods The biological characteristics of Hep3B cells,such as proliferation,migration and invasion,were comparatively analyzed under HUVECs-derived conditioned medium (CM) stimulation and endothelial cell basal medium (EBM) stimulation,and its related signal molecules were detected by Western blotting.Data analysis was performed using SPSS 16.0 software.Results Cell Counting Kit-8 (CCK-8) test showed that the proliferation of Hep3 B cells treated with CM was significantly increased as compared with that of the control cells (P ＜ 0.01).The wound healing assay and cell migration assay demonstrated that the migration of Hep3B cells was obviously enhanced underCM stimulation [(33.65±1.22)％ vs.(57.85±2.20)％,P＜0.01 and 51.2±3.1 vs.28.4± 4.6,P ＜0.01],and invasion ability of Hep3B cells induced by CM was also improved (16.2 ± 2.3 vs.5.4 ± 1.5).The expressions of invasion/metastasis associated genes matrix metalloproteinase-2 (MMP-2),matrix metalloproteinase-9 (MMP-9),osteopontin (OPN) and cluster of differentiation (CD44) and the serine-threonine kinase (Akt) phosphorylation and Extracellular Regulated Protein kinases (ERK) phosphorylation level were all upregulated in the treated Hep3B with CM.Conclusion Vascular endothelial cell conditioned mediun containing some soluble cytokines and/or chemokines which may facilitate the proliferation,migration and invasion of HCC cells via paracrine pathway.