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目的组织工程骨的神经化能有效促进支架材料内血管生成,修复骨缺损。研究降钙素基因相关肽(calcitonin gene-related peptide,CGRP)对人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖与迁移的作用,进一步揭示组织工程骨的神经化促血管生成机制。方法体外分离获取HUVECs,并通过血管性血友病因子(von Willebrand factor,vWF)与CD31抗原鉴定,取第1代细胞用于实验。实验分为6组,分别以0(A组)、1×10—12(B组)、1×10—11(C组)、1×10—10(D组)、1×10—9(E组)、1×10—8mol/L(F组)浓度CGRP干预HUVECs。采用细胞免疫荧光观察HUVECs的CGRP1受体(CGRP1 receptor,CGRP1R)表达情况,AlarmarBlue法动态检测各组HUVECs增殖率,Transwell小室检测各组HUVECs的迁移能力,ELISA法检测HUVECs分泌VEGF的水平,Westernblot法检测其局部黏着斑激酶(focal adhesion kinase,FAK)的表达。结果分离的细胞通过形态学及vWF、CD31免疫荧光鉴定为HUVECs,并可见CGRP1R在细胞质和细胞膜表达。CGRP呈时间-浓度依赖性刺激HUVECs增殖;B～F组各时间点细胞增殖能力均高于A组(P<0.05),F组各时间点细胞增殖能力最高。B～F组迁移细胞数均显著高于A组(P<0.05),最大增幅达3倍以上。B～F组VEGF分泌量均显著高于A组(P<0.05);C、D组促进细胞分泌VEGF的能力最强。Western blot检测示,与A组相比,B～F组CGRP刺激HUVECs 3、7、10 d后,FAK表达明显增加(P<0.05)。结论 CGRP对HUVECs的增殖和迁移有直接促进作用,可能作用机制为CGRP能促进VEGF分泌和增加FAK的表达。
Objective The neurogenicization of tissue engineered bone can effectively promote angiogenesis in scaffolds and repair bone defects. To investigate the effect of calcitonin gene-related peptide (CGRP) on the proliferation and migration of human umbilical vein endothelial cells (HUVECs) and to further reveal the mechanism of neurogenic angiogenesis in tissue engineered bone . Methods HUVECs were obtained in vitro and identified by von Willebrand factor (vWF) and CD31 antigen. The first generation of cells were used for the experiment. The experiment was divided into six groups, which were divided into four groups: 0 (group A), 1 × 10-12 (group B), 1 × 10-11 (group C), 1 × 10-10 (group D) E group) and 1 × 10-8mol / L (F group) CGRP intervention HUVECs. The expression of CGRP1 receptor (CGRP1R, CGRP1R) in HUVECs was observed by cell immunofluorescence. The proliferation rate of HUVECs in each group was detected by AlarmarBlue method. The migration ability of HUVECs in each group was detected by Transwell chamber. The level of VEGF secreted by HUVECs was detected by ELISA. To detect the expression of focal adhesion kinase (FAK). Results The isolated cells were identified as HUVECs by morphological, vWF and CD31 immunofluorescence. The expression of CGRP1R in cytoplasm and cell membrane was also observed. CGRP stimulated the proliferation of HUVECs in a time-and-concentration-dependent manner; the cell proliferation ability of B ~ F group was higher than that of A group at each time point (P <0.05); The number of migrated cells in B ~ F group was significantly higher than that in A group (P <0.05), and the maximum increase was more than 3 times. The VEGF secretion of B ~ F group was significantly higher than that of A group (P <0.05). C and D groups had the strongest ability of promoting VEGF secretion. Western blot showed that compared with group A, FAK expression in HUVECs was significantly increased (P <0.05) after CGRP stimulated in B ~ F group at 3, 7 and 10 d. Conclusions CGRP can directly promote the proliferation and migration of HUVECs. The possible mechanism is that CGRP can promote the secretion of VEGF and increase the expression of FAK.