胶质瘤细胞与血管内皮细胞的信号Crosstalk对肿瘤细胞增殖和侵袭的影响

Effects of signal Crosstalk between glioma cells and vascular endothelial cells on the proliferation and invasion of glioma cells

目的探讨胶质瘤细胞与血管内皮细胞之间的信号Crosstalk及其对肿瘤细胞增殖和侵袭的影响。方法将人脑微血管内皮细胞(HBMEC)分4组培养:对照组为HBMEC常规培养,血管内皮生长因子(VEGF)组为HBMEC在含VEGF_(165)(50 ng/mL)的内皮细胞培养基(ECM)中培养,U87MG共培养组为HBMEC与胶质瘤细胞U87MG共培养,U251共培养组为HBMEC与胶质瘤细胞U251共培养,然后采用ELISA检测胶质瘤细胞及组织因子VEGF对血管内皮细胞分泌CXCL8的影响。将人脑胶质瘤细胞U87MG或U251分4组培养:对照组为U87MG或U251常规培养,U87MG/U251+HBMEC组为U87MG或U251与HBMEC共培养,U87MG/U251+HBMEC+rhCXCL8组为U87MG或U251与HBMEC在含Akt通路的激动剂rhCXCL8(50 ng/mL)的DMEM中共培养,U87MG/U251+HBMEC+LY294002组为U87MG或U251与HBMEC在含Akt通路的抑制剂LY294002(1μmol/L)的DMEM中共培养,分别采用Western blotting、CCK-8、Transwell侵袭实验检测血管内皮细胞及其分泌CXCL8对胶质瘤细胞的增殖、侵袭以及Akt蛋白表达的影响。结果与对照组比较,VEGF组、U87MG共培养组、U251共培养组中HBMEC培养体系的CXCL8分泌量升高(P<0.05),胶质瘤细胞及其分泌VEGF可促进血管内皮细胞分泌CXCL8。与对照组比较,U87MG/U251+HBMEC组细胞P-Akt蛋白表达增高(P<0.05),增殖及侵袭能力增高(P<0.05);给予CXCL8激活使P-Akt蛋白表达、增殖及侵袭进一步升高(P<0.01);给予LY294002抑制则P-Akt的表达降低(P<0.01),血管内皮细胞可通过分泌CXCL8激活胶质瘤细胞Akt信号通路,并促进其增殖和侵袭。结论胶质瘤细胞与血管内皮细胞之间存在VEGF-CXCL8-Akt信号Crosstalk机制,在胶质瘤增殖和侵袭中发挥重要作用。

Objective To explore the signal Crosstalk between glioma cells and human brain microvascular endothelial cells(HBMECs)and its effects on the proliferation and invasion of glioma cells.Methods HBMECs were divided into four groups:the control group was cultured with conventional endothelial cell medium(ECM);the vascular endothelial growth factor(VEGF)group was cultured with ECM treated with VEGF165(50 ng/mL);U87 MG co-culture group was co-cultured with U87 MG cells;U251 co-culture group was co-cultured with U251 cells.Then ElISA was performed to detect the effects of glioma cells and VEGF on the secretion of CXCL8.The glioma cells(U87 MG/U251)were divided into four groups:the control group was cultured with conventional DMEM;U87 MG/U251+HBMEC group was co-cultured with HBMECs,U87 MG/U251+HBMEC+rhCXCL8 group was co-cultured with HBMECs in DMEM containing rhCXCL8(50 ng/mL,Akt pathway agonist),U87 MG/U251+HBMEC+LY29400 group were co-cultured with HBMECs in DMEM containing LY294002(1μmol/L,Akt pathway inhibitor).Then Western blotting,CCK-8 and Transwell assays were performed to examine the effects of HBMECs and secretion of CXCL8 on the proliferation and invasion of glioma cells and expression of Akt.Results The secretion of CXCL8 in the VEGF group,U87 MG co-culture group and U251 co-culture group were significantly increased compared with that in the control group(P<0.05),which indicated that glioma cells and VEGF could promote the secretion of CXCL8.Compared with the control group,the U87 MG/U251+HBMEC group had elevated P-Akt,proliferation and invasion(P<0.05);exposure to CXCL8 further increased P-Akt,proliferation and invasion(P<0.01),whereas exposure to LY294002 suppressed P-Akt(P<0.01),which indicated that HBMECs could activate Akt signaling pathway by secreting CXCL8,and promote the proliferation and invasion of glioma cells.Conclusion There is a crosstalk of VEGF-CXCL8-Akt signal network between glioma cells and HBMECs,which might play an important role in the proliferation and invasion of glioma cells.