降钙素基因相关肽对血管内皮细胞体外血管生成的作用及机制研究

Effect of calcitonin gene-related peptide on angiogenesis of human umbilical vein endothelial cells

目的观察降钙素基因相关肽（calcitonin-gene-related peptide，CGRP）对人脐静脉血管内皮细胞human umbilical vein endothehal cells，HUVECs）的成血管作用，初步探讨其在骨组织工程中的应用价值。方法体外分离获取HUVECs，采用细胞免疫荧光检测其CGRP受体1的表达。体外成管实验检测CGRP的成血管作用．ELISA法检测CGRP直接作用于HUVECs时血管内皮生长因子vascular endothelial growth factor，VEGF）的分泌水平。Q—PCR检测CGRP刺激HUVECs不同时间点VEGF、VEGF受体l（FLT1）、VEGF受体2（KDR）及CGRP受体lmRNA的表达，Westernblot检测HUVECs不同时间点FLT1、KDR的蛋白表达。结果细胞免疫荧光显示HUVECs表达CGRP受体1，体外成管实验显示CGRP有明显的成血管作用。ELISA显示CGRP能明显促进HUVEC分泌VEGF。Q—PCR结果显示不同浓度组CGRP受体1mRNA的表达较对照组增高，且在第10天最为明显；Q—PCR及Westernblot结果显示不同浓度组FIJT1、KDR mRNA和蛋白的表达在各时间点较对照组均增高。结论CGRP能明显促进HUVECs的体外生成血管，可能与其促进VEGF分泌，增强HUVECs的FLTl与KDR表达有关；同时，CGRP受体表达也增加，可进一步增强CGRP的促血管生成作用。

Objective To investigate the effect of calcitonin gene-related peptide （CGRP） on angiogenesis of human umbilical vein endothelial cells （I-IUVECs）. Methods The HUVECs were collected from human umbilical core, and the expression of the CGRP receptor-1 was identified though immunofluorescence. After HUVECs were treated with CGRP, the angiogenesis was detected through tube formation experiment. The secretion of vascular endothelial growth factor （VEGF） was detected through ELISA method. The mRNA expression of VEGF, VEGF receptor-1 （FLT1）, VEGF receptor-2 （KDR） and CGRP receptor-1 were detected through quantitative PCR （Q-PCR） at 3, 7, 10 days after culturing. Western blot method was used to detect the protein expression of FLT1 and KDR in HUVECs. Results Immunofluorescence result showed CGRP receptor-1 expressed in HUVECs. CGRP could significantly promote angiogenesis and increase VEGF secretion in direct manner. The Q-PCR resuhs showed that the mRNA expression level of CGRP receptor-1 was significantly higher in CGRP groups than that in control group, especially at 10 days. Compared to the control group, the mRNA and protein expression level of FLT1 and KDR were statistically higher in CGRP groups at different time. Conclusion CGRP can significantly promote angiogenesis of HUVECs in vitro, which may be because it can promote VEGF secretion and expression of FLT1 and KDR in HUVECs. Meanwhile, the increase of CGRP receptor-1 expression also can promote angiogenesis of HUVECs.