降钙素基因相关肽对人脐静脉血管内皮细胞增殖和迁移的影响及机制研究

EFFECT OF CALCITONIN GENE-RELATED PEPTIDE ON PROLIFERATION AND MIGRATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS

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 Tissue engineered bone implanted with sensory nerve can effectively promote angiogenesis and repair of bone defects.To investigate the effects of calcitonin gene-related peptide（CGRP） on proliferation and migration of human umbilical vein endothelial cells（HUVECs） for further revealing the mechanism of tissue engineered bone implanted with sensory nerve promoting angiogenesis.Methods HUVECs were collected from human umbilical core,and identified through von Willebrand factor（vWF） and CD31 immunofluorescence.The HUVECs were treated with CGRP and were divided into 6 groups according to CGRP concentration： group A（0 mol/L）,group B（1 × 10—12 mol/L）,group C（1 × 10—11 mol/L）,group D（1 × 10—10 mol/L）,group E（1 × 10—9 mol/L）,and group F（1 × 10—8 mol/L）.The expression of the CGRP1 receptor（CGRP1R） was observed in HUVECs by cell immunofluorescence.The growth rate of HUVECs was detected through AlarmarBlue at 1,2,3,4,and 5 days.Transwell chamber was used to detect the ability of cell migration.ELISA assay was used to detect the vascular endothelial growth factor（VEGF） secretion and the protein expression of focal adhesion kinase（FAK） was examined using Western blot.Results HUVECs were identified through morphology,vWF and CD31 immunofluorescence.HUVECs expressed CGRP1R.CGRP could stimulate HUVECs proliferation in a time-and concentration-dependent manners;the cell growth rates of groups B-F were significantly higher than that of group A at all time（P 0.05）;group F had highest cell growth rate.The number of cell migration of group B-F was significantly higher than that of group A（P 0.05）,which increased more than 3 times.Groups B-F had higher amount of VEGF than group A（P 0.05）,and groups C and D had highest amount of VEGF.FAK expression of groups B-F was significantly increased at 3,7,and 10 days after CGRP treatment when compared with group A（P 0.05）.Conclusion CGRP may enhance the proliferation and migration of HUVECs by increasing the secretion of VEGF and expression of FAK.