氯化钴诱导人肝窦内皮细胞缺氧模型特点

Characteristics of hypoxia model induced by cobalt chloride in human hepatic sinusoidal endothelial cells

目的观察氯化钴(CoCl_2)诱导人肝窦内皮细胞(HHSEC)缺氧模型特点。方法不同浓度CoCl2(0~800μmol/L)与HHSEC细胞孵育24 h,CCK8法检测细胞活力;50~200μmol/L CoCl2与HHSEC细胞孵育,Transwell实验检测细胞迁移;Matrigel管腔生成实验检测细胞管腔形成情况;蛋白质免疫检测细胞HIF-1α、vWF、VEGF蛋白表达;免疫荧光染色观察HIF-1α核转位。结果与对照组比较,50~200μmol/L CoCl2组HHSEC细胞活力明显增加,细胞迁移到膜下的数量显著增多,并且形成丰富而密闭的管状结构。50μmol/L、200μmol/L CoCl2促进HHSEC中缺氧诱导因子1α(HIF-1α)、第八因子相关抗原(vWF)、血管内皮生长因子(VEGF)表达,其中200μmol/L CoCl2组HIF-1α、vWF表达较50μmol/L CoCl2组高。结论 50μmol/L、200μmol/L低浓度CoCl2可诱导人肝窦内皮细胞缺氧、血管新生,其作用机制与促进HIF-1α表达及核转位相关。

Objective To investigate the characteristics of hypoxia model induced by cobalt chloride(CoCl 2)in human hepatic sinusoidal endothelial cells(HHSEC).Methods HHSEC were incubated with different concentrations of CoCl 2(0~800μM)for 24 h,in which cell viability was measured with counting kit-8(CCK8)assay.Cell migration was detected by transwell assay after stimulating HHSEC with 50~200μM CoCl 2.Matrigel lumen formation assay was used to detect cell lumen formation.Expressions of cell hypoxia inducible factor-1α(HIF-1α),von Willebrand factor(vWF)and vascular endothelial growth factor(VEGF)protein were detected by western blot.HIF-1αnuclear translocation was observed by immunofluorescence staining.Results Compared with control group,cell viability and cell migration of HHSEC were significantly increased,with a rich and airtight tubular structure formed in low concentration of 50~200μM CoCl 2 group.Expressions of HIF-1α,vWF and VEGF in HHSEC were increased in 50μM and 200μM CoCl 2 group than those in the control group.Furthermore,expressions of HIF-1αand vWF in 200μM CoCl 2 group were higher than those in 50μM CoCl 2 group.Conclusion Low levels of 50μM and 200μM CoCl 2 may induce HHSEC hypoxia and angiogenesis through promoting the expression and nuclear translocation of HIF-1α.