肿瘤内皮来源CXC趋化因子配体8对肝细胞癌血管生成拟态的影响

Effects of tumor endothelial cells derived CXC chemokine ligand 8 on vasculogenic mimicry of hepatocellular carcinoma

目的探讨肿瘤内皮细胞(TECs)来源CXC趋化因子配体8(CXCL8)在肝细胞癌(HCC)血管生成拟态(VM)中的作用。方法收集2018年9月到2019年9月在北京朝阳医院行手术切除的HCC组织,免疫磁珠法分离TECs;慢病毒感染TECs,敲低其CXCL8表达,分为对照组和敲低组,收集其条件培养基;酶联免疫吸附试验检测TECs条件培养基中CXCL8的表达;小管形成实验观察HCC细胞VM;蛋白质印迹法观察HCC细胞中相关蛋白水平变化。组间比较采用独立样本t检验或单因素方差分析。结果TECs敲低组中CXCL8蛋白表达水平较对照组明显降低。对照组条件培养基中CXCL8水平[(1076.00±88.80)pg/ml]较敲低组明显升高[(787.50±63.43)pg/ml],差异有统计学意义(t=5.920,P<0.01)。对照组和敲低组条件培养基处理HepG2细胞后相对小管长度分别为(9814.0±692.8)和(6434.0±1272.0),差异有统计学意义(t=4.042,P<0.05);在SMMC7721细胞中,对照组和敲低组分别为(22317.0±3121.0)和(9834.0±1534.0),差异有统计学意义(t=6.217,P<0.01)。CXC趋化因子受体1(CXCR1)中和性抗体预处理HCC细胞后,TECs促进HCC细胞的VM能力减弱。对照组条件培养基较敲低组明显促进HCC细胞中波形蛋白(Vimentin)和Snail的表达。HepG2细胞中Vimentin的表达量为0.178±0.020和0.094±0.023(t=4.707,P<0.01),Snail的表达量为0.273±0.020和0.188±0.033(t=3.755,P<0.05);SMMC7721细胞中Vimentin的表达量为0.342±0.020和0.242±0.035(t=4.270,P<0.05),Snail的表达量为0.189±0.014和0.139±0.021(t=3.354,P<0.05)。结论TECs通过旁分泌CXCL8方式结合HCC细胞表面CXCR1,调节上皮-间充质转化(EMT)来促进HCC细胞VM。

Objective To explore the effect of tumor endothelial cells(TECs)derived CXC chemokine ligand 8(CXCL8)on vasculogenic mimicry(VM)in hepatocellular carcinoma(HCC)cells.Methods Resected HCC tissued were collected in Beijing Chaoyang Hospital from September 2018 to September 2019.Immunomagnetic bead methods were used to separate TECs.Lentivirus vectors were used to knock down the expression of CXCL8 in TECs.The groups were divided into control group and knockdown group.VM was observed by tube formation.CXCL8 in conditioned medium was measured by Enzyme-linked immunosorbent assay.Western blotting was used to examine the associated proteins in HCC.Independent samples t test or one-way ANOVA was used in statistical analysis.Results Control group showed higher expression of CXCL8 compared with knockdown group.Higher levels of CXCL8 in conditioned media were found in control group[(1076.00±88.80)pg/ml]compared with that in knockdown group[(787.50±63.43)pg/ml,t=5.920,P<0.01].Conditioned media from control group could significantly promote VM compared with that from knockdown group[HepG2:(9814.0±692.8)vs.(6434.0±1272.0),t=4.042,P<0.05;SMMC7721:(22317.0±3121.0)vs.(9834.0±1534.0),t=6.217,P<0.01].However,after pretreating HCC cells using neutralizing antibody of CXC chemokine receptor 1(CXCR1),the ability of conditioned media promoting VM in HCC cells was decreased.Conditioned media from control group could significantly increase the expression of Vimentin and Snail compared with that from knockdown group in HCC cells.The relative expression of Vimentin were 0.178±0.020 and 0.094±0.023(t=4.707,P<0.01),and the relative expression of Snail were 0.273±0.020 and 0.188±0.033(t=3.755,P<0.05)in HepG2 cells;the relative expression of Vimentin were 0.342±0.020 and 0.242±0.035(t=4.270,P<0.05),and the relative expression of snail were 0.189±0.014 and 0.139±0.021(t=3.354,P<0.05)in SMMC7721 cells.Conclusion TECs promotes VM of HCC cells by a paracrine manner through CXCL8/CXCR1/Snail/Vimentin pathways.