MDA-MB-231细胞源exosome对人脐静脉内皮细胞(HUVEC)VEGF自分泌及体外成管作用的影响

 目的 研究人乳腺癌MDA MB 231细胞源exosome对人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)血管内皮生长因子(vascular endothelial growth factor,VEGF)自分泌及体外成管作用的影响,探讨肿瘤细胞源exosome在肿瘤微环境中对血管内皮细胞血管生成的调控作用。方法 低温超速离心及密度梯度离心法提取乳腺癌MDA MB 231细胞源exosome;酶联免疫吸附试验(ELISA)检测HUVEC与exosome共培养24 h后上清液中VEGF的变化水平;Western blot技术检测HUVEC与exosome共培养24 h后VEGF、VEGFR2及p VEGFR2的蛋白表达情况;RT PCR法检测HUVEC与exosome共培养24 h后VEGF的基因表达情况;观察HUVEC与exosome共培养24 h后的体外成管能力。 结果 HUVEC与exosome共培养24 h后上清液中VEGF为(110.851±18.404)pg/mL,与对照组相比差异具有统计学意义(P＜0.05);Western blot结果显示,HUVEC与exosome共培养24 h后VEGF和p VEGFR2的蛋白表达水平均增加(P＜0.05);RT PCR结果显示,HUVEC与exosome共培养24 h后VEGF的基因表达水平增加(P＜0.05);体外成管实验显示,exosome显著提高了HUVEC的管腔形成能力(P<0.05)。结论 乳腺癌MDA MB 231细胞源exosome促进了血管内皮细胞VEGF的表达及分泌,激活了血管内皮细胞VEGF/VEGFR2自分泌环并提高了血管内皮细胞的体外成管能力,对促肿瘤血管生成有一定的调控作用。

Effects of exosomes derived from MDA-MB-231 on the expression of autocrine VEGF and capillary-like tube formation in HUVECs

Objective To investigate the effects of exosomes derived from breast cancer cell line MDA MB 231 on the expression of autocrine vascular endothelial growth factor (VEGF) and capillary like tube formation in human umbilical vein endothelial cells (HUVECs),and to observe the regulatory effect of exosomes derived from cancer cells on angiogenesis in tumor microenvironment.Methods Exosomes were purified by serial ultracentrifugation and sugar density ultracentrifugation.The expression of autocrine VEGF in HUVECs with exosomes co cultured 24 hours were detected by ELISA.The expressions of VEGF,VEGFR2 and p VEGFR2 in HUVECs co cultured with exosomes for 24 hours were detected by Western blot.RT PCR was used to detect the mRNA expression of VEGF in HUVECs co cultured with exosomes for 24 hours.The capillary like tube formations by HUVECs co cultured with exosomes for 24 hours were observed.Results ELISA result showed that the concentration of VEGF in HUVECs co cultured with exosomes for 24 hours was (110.851±18.404) pg/mL,which was significantly different from the untreated HUVECs (P＜0.05).Western blot result showed that the expressions of VEGF and p VEGFR2 in HUVECs co cultured with exosomes for 24 hours were significantly higher than those in the untreated HUVECs (P＜0.05).RT PCR result showed that the mRNA expression of VEGF in HUVECs co cultured with exosomes for 24 hours was significantly different from the untreated HUVECs (P＜0.05).Conclusions Exosomes derived from breast cancer cell line MDA MB 231 can promote the expression of autocrine VEGF,the activation of VEGF/VEGFR2 autocrine loop and the capability of capillary like tube formation of HUVECs.They may be associated with the regulation in tumor angiogenesis.