中华骨科杂志
中華骨科雜誌
중화골과잡지
CHINESE JOURNAL OF ORTHOPAEDICS
2012年
8期
781-787
,共7页
陀泳华%郭小磊%张鑫鑫%王钊%文军%周健%夏立恒%张永涛%金丹
陀泳華%郭小磊%張鑫鑫%王釗%文軍%週健%夏立恆%張永濤%金丹
타영화%곽소뢰%장흠흠%왕쇠%문군%주건%하립항%장영도%금단
降钙素基因相关肽%脐静脉%内皮细胞%组织工程%骨
降鈣素基因相關肽%臍靜脈%內皮細胞%組織工程%骨
강개소기인상관태%제정맥%내피세포%조직공정%골
Calcitonin gene-related peptide%Umbilical veins%Endothelial cells%Tissue engineering%Bones
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 endothelial 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l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1(FLT1)、VEGF受体2(KDR)及CGRP受体1 mRNA的表达,Western blot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1 mRNA的表达较对照组增高,且在第10天最为明显;Q-PCR及Western blot结果显示不同浓度组FLT1、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FLT1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.
目的 觀察降鈣素基因相關肽(calcitonin-gene-related peptide,CGRP)對人臍靜脈血管內皮細胞(human umbilical vein endothelial cells,HUVECs)的成血管作用,初步探討其在骨組織工程中的應用價值.方法 體外分離穫取HUVECs,採用細胞免疫熒光檢測其CGRP受體l的錶達.體外成管實驗檢測CGRP的成血管作用,ELISA法檢測CGRP直接作用于HUVECs時血管內皮生長因子(vascular endothelial growth factor,VEGF)的分泌水平.Q-PCR檢測CGRP刺激HUVECs不同時間點VEGF、VEGF受體1(FLT1)、VEGF受體2(KDR)及CGRP受體1 mRNA的錶達,Western blot檢測HUVECs不同時間點FLT1、KDR的蛋白錶達.結果 細胞免疫熒光顯示HUVECs錶達CGRP受體1,體外成管實驗顯示CGRP有明顯的成血管作用.ELISA顯示CGRP能明顯促進HUVEC分泌VEGF.Q-PCR結果顯示不同濃度組CGRP受體1 mRNA的錶達較對照組增高,且在第10天最為明顯;Q-PCR及Western blot結果顯示不同濃度組FLT1、KDR mRNA和蛋白的錶達在各時間點較對照組均增高.結論 CGRP能明顯促進HUVECs的體外生成血管,可能與其促進VEGF分泌,增彊HUVECs的FLT1與KDR錶達有關;同時,CGRP受體錶達也增加,可進一步增彊CGRP的促血管生成作用.
목적 관찰강개소기인상관태(calcitonin-gene-related peptide,CGRP)대인제정맥혈관내피세포(human umbilical vein endothelial cells,HUVECs)적성혈관작용,초보탐토기재골조직공정중적응용개치.방법 체외분리획취HUVECs,채용세포면역형광검측기CGRP수체l적표체.체외성관실험검측CGRP적성혈관작용,ELISA법검측CGRP직접작용우HUVECs시혈관내피생장인자(vascular endothelial growth factor,VEGF)적분비수평.Q-PCR검측CGRP자격HUVECs불동시간점VEGF、VEGF수체1(FLT1)、VEGF수체2(KDR)급CGRP수체1 mRNA적표체,Western blot검측HUVECs불동시간점FLT1、KDR적단백표체.결과 세포면역형광현시HUVECs표체CGRP수체1,체외성관실험현시CGRP유명현적성혈관작용.ELISA현시CGRP능명현촉진HUVEC분비VEGF.Q-PCR결과현시불동농도조CGRP수체1 mRNA적표체교대조조증고,차재제10천최위명현;Q-PCR급Western blot결과현시불동농도조FLT1、KDR mRNA화단백적표체재각시간점교대조조균증고.결론 CGRP능명현촉진HUVECs적체외생성혈관,가능여기촉진VEGF분비,증강HUVECs적FLT1여KDR표체유관;동시,CGRP수체표체야증가,가진일보증강CGRP적촉혈관생성작용.
Objective To investigate the effect of calcitonin gene-related peptide (CGRP) on angiogenesis of human umbilical vein endothelial cells (HUVECs).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 results 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.