吗啡对人肝癌细胞Huh7诱导HUVECs血管生成能力的影响及其分子机制

Effect of morphine on human hepatocellular carcinoma Huh7 cells-induced angiogenesis of HUVECs and its molecular mechanism

目的观察吗啡对人肝癌细胞Huh7诱导人脐静脉内皮细胞(HUVECs)血管生成能力的影响,并探讨其可能的分子机制。方法取对数生长期Huh7细胞,加入不同浓度吗啡(0、0.01、0.1、1、10、100、1000μmol/L)作用不同时间(0、6、12、24 h),根据细胞增殖率及细胞毒性检测结果选择1μmol/L吗啡作用24 h为最佳条件。将Huh7细胞分为观察组和对照组,观察组加入1μmol/L吗啡作用24 h,对照组不予特殊处理。收集两组上清液,与ECM基础培养液混合后孵育HUVECs,采用管腔形成实验观察其血管生成能力(以管腔形成数量表示);采用Western blotting法检测细胞缺氧诱导因子1α(HIF-1α)蛋白表达,ELISA法检测上清液中血管内皮生长因子(VEGF)表达。将Huh7细胞分为si-HIF-1α组、si-NC组和空白对照组,si-HIF-1α组、si-NC组分别采用脂质体Lipofectamin TM 2000法转染siHIF-1α及其阴性对照,空白对照组不予特殊处理,各组均加入1μmol/L吗啡作用24 h,比较各组血管生成能力及HIF-1α、VEGF表达。结果与对照组比较,观察组管腔形成数量及HIF-1α、VEGF表达均升高(P均<0.05)。与si-NC组、空白对照组比较,si-HIF-1α组管腔形成数量及HIF-1α、VEGF表达均降低(P均<0.05)。si-NC组与空白对照组管腔形成数量及HIF-1α、VEGF表达比较均无统计学差异(P均>0.05)。结论吗啡可促进Huh7细胞诱导HUVECs血管生成,其机制可能与激活HIF-1α表达、促进VEGF分泌有关。

Objective To investigate the effect and molecular mechanism of morphine on human hepatocellular carcinoma Huh7 cells-induced angiogenesis of human umbilical vascular endothelial cells(HUVECs).Methods After Huh7 cells in the logarithmic phase were exposed to morphine at different concentrations(0.01,0.1,1,10,100,and 1000μmol/L)and different time points(6,12,and 24 h),the ability of cell proliferation and the cytotoxicity to morphine were examined by CCK-8 and LDH release assays,respectively.Based on the two results,Huh7 cells cultured in 1μmol/L morphine for 24 h were the optimum condition for next experiments.Huh7 cells were randomly divided into two groups:the observation group treated with 1μmol/L morphine for 24 h and the control group without morphine.HUVECs were cultured in the tumor conditioned medium derived from supernatant fluid of Huh7 cells,and lumen formation assay was used to assess the ability of HUVECs tube formation(the number of lumen formation of HUVECs).The expression of hypoxia-inducible factor-1α(HIF-1α)of Huh7 cells and the concentration of vascular endothelial growth factor(VEGF)from supernatant fluid were determined by Western blotting and ELISA,respectively.Huh7 cells were randomly assigned into three groups:the si-HIF-1αgroup,si-con group,and NC group;cells in the si-HIF-1αgroup and si-con group were separately transfected with HIF-1αsmall interfering RNA and disordered nonsense sequence,but cells in the NC group were not treated.After Huh7 cells with si-RNA transfection were treated with morphine for 24 h,the ability of HUVECs tube formation,as well as the expression of HIF-1αand VEGF was examined with experiments mentioned above.Results Compared with the control group,the number of tube formation,and the expression of HIF-1αand VEGF increased in supernatant fluid of the observation group(all P<0.05).Compared with the si-con group and control group,the number of tube formation,and the expression of HIF-1αand VEGF decreased in the siRNA-HIF-1αgroup(all P<0.05).Furthermore,there was no significant difference between the si-con group and control group(all P>0.05).Conclusions Morphine can indirectly stimulate the lumen formation of HUVECs via activating the expression of HIF-1αand promoting the secretion of VEGF.