肿瘤内皮细胞来源CXC趋化因子配体8募集巨噬细胞对人肝癌细胞Hep3B增殖和侵袭能力的影响

Effect of recruitment of macrophages by CXC chemokine ligand 8 from tumor endothelial cells on proliferation and invasion of hepatocellular carcinoma Hep3B cells

目的:探讨肿瘤内皮细胞(TECS)来源的CXC趋化因子配体8(CXCL8)募集巨噬细胞在人肝癌细胞Hep3B增殖和侵袭中的作用。方法:分离和培养TECS。诱导THP-1细胞为巨噬细胞。Hep3B和THP-1细胞购自国家实验细胞资源共享平台,TECs提取自2022年9月至2023年9月在北京朝阳医院行手术切除的HCC组织。收集TECS条件培养基、巨噬细胞条件培养基及TECS和巨噬细胞共培养条件培养基。细胞计数试剂盒(CCK-8)增殖实验观察上述条件培养基对Hep3B细胞增殖能力的改变。Transwell侵袭实验检测该条件培养基对Hep3B细胞侵袭能力的改变。酶联免疫吸附实验检测条件培养基中CXCL8和血管内皮生长因子A(VEGFA)的浓度。独立样本 t检验或单因素方差分析比较两组或3组间的差异。 结果:TECS组巨噬细胞迁移高于对照组(411.70±34.49比211.30±36.12, t=6.949, P<0.01),条件培养基中加入CXCL8中和性抗体组巨噬细胞迁移低于对照IgG抗体组(189.30±8.02比376.00±35.55, t=8.871, P<0.01)。TECS和巨噬细胞共培养的条件培养基组Hep3B细胞增殖和侵袭均高于TECS条件培养基组和巨噬细胞条件培养基组,细胞增殖72小时OD值分别为(2.468±0.093比1.878±0.163、1.946±0.120, F= 37.710, P<0.01);细胞侵袭数分别为(535.00±62.38比403.70±26.08、385.00±53.33, F=8.111, P<0.05)。TECS和巨噬细胞共培养的条件培养基组中VEGFA的浓度高于TECS条件培养基组和巨噬细胞条件培养基组(1 362.00±106.80比651.00±52.65、749.20±52.98, F=157.300, P<0.01);TECS和巨噬细胞共培养条件培养基加入VEGFA中和性抗体组Hep3B增殖和侵袭均低于IgG对照组,细胞增殖72小时OD值分别为(1.146±0.057比1.802±0.047, t=21.700, P<0.01)细胞侵袭数分别为(230.70±75.95比499.70±60.71, t=4.791, P<0.01)。 结论:TECS通过CXCL8促进巨噬细胞迁移,进而通过VEGFA促进人肝癌细胞Hep3B细胞的增殖和侵袭。

Objective:To explore the effect of tumor endothelial cells(TECS)derived CXC chemokine ligand 8(CXCL8)on proliferation and invasion in hepatocellular carcinoma(HCC)cells.Methods:TECS were separated and cultured.THP-1 cells were induced into macrophages.Hep3B and THP-1 cells were purchased from the National Experimental Cell Resource Sharing Platform,and TECs were extracted from HCC tissues surgically resected at Beijing Chaoyang Hospital from September 2022 to September 2023.Collect the conditioned medium from TECS,macrophages and TECS+macrophages.Cell counting kit-8(CCK-8)proliferation experiment was used to examine the proliferation of Hep3B cells.Transwell invasion experiment was used to examine the invasion of Hep3B cells.Enzyme-linked immunosorbent assay was used to examine the concentration of vascular endothelial growth factor A(VEGFA)in the conditioned medium.Independent samples t test or one-way ANOVA was used in statistical analysis.Results:The TECS group significantly promoted the migration of macrophages compared with the control group(411.70±34.49 vs.211.30±36.12,t=6.949,P<0.01),and the addition of CXCL8 antibody to the conditioned medium significantly inhibited the migration of macrophages compared with the control IgG group(189.30±8.02 vs.376.00±35.55,t=8.871,P<0.01).The proliferation and invasion of Hep3B cells in conditioned medium co-cultured with TECS and macrophages group were higher compared with TECs group and macrophage group,the 72 h OD values were(2.468±0.093 vs.1.878±0.163,1.946±0.120,F=37.710,P<0.01)and the invasion numbers were(535.00±62.38 vs.403.70±26.08,385.00±53.33,F=8.111,P<0.05)of Hep3B cells compared with the conditioned medium from TECS or macrophages.The concentration of VEGFA in the conditioned medium of co-culture of TECS and macrophages was significantly increased compared with the conditioned medium of TECS and the conditioned medium of macrophages(1362.00±106.80 vs.651.00±52.65,749.20±52.98,F=157.300,P<0.01).The proliferation and invasion of Hep3B cells in conditioned medium blocking VEGFA with VEGFA neutralizing antibody group were lower compared with IgG group,the 72 h OD values were(1.146±0.057 vs.1.802±0.047,t=21.700,P<0.01)and the invasion numbers were(230.70±75.95 vs.499.70±60.71,t=4.791,P<0.01).Conclusion:TECS recruit macrophages through CXCL8,and promote the proliferation and invasion of Hep3B cells through VEGFA.