蛇毒金属蛋白酶抑制剂重组蛋白抗肿瘤血管生成作用及其机制  

The inhibition effects and mechanism of recombinant snake venom metalloproteinase inhibitor on tumor angiogenesis

作　　者：纪明开[1,2] 陈丽红[1] 程波[1] 师仪 林旭[2] 林建银[2] 

机构地区：[1]福建医科大学附属第一医院皮肤科,福建福州350004 [2]福建医科大学分子医学研究中心消化道恶性肿瘤教育部重点实验室,福建福州350004 [3]福建省肿瘤医院病理科,福建福州350014

出　　处：《中国药理学通报》2017年第3期394-400,共7页Chinese Pharmacological Bulletin

基　　金：福建省卫生系统中青年骨干人才培养项目(No 2014-ZQNJC-17)

摘　　要：目的研究蛇毒金属蛋白酶抑制剂重组蛋白(recombinant snake venom metalloproteinase inhibitor,r SVMPI)对血管生成的影响及其分子机制。方法应用鸡胚绒毛尿囊膜(chicken chorioallantoic membrane,CAM)血管生成模型观察SVMPI重组蛋白对血管生成的影响;采用Alamar Blue分析方法检测人脐静脉内皮细胞(human umbilical veins endothelial cells,HUVECs)增殖能力、Annexin V-FITC双标记流式细胞术检测细胞凋亡、划痕标记法检测细胞体外迁移能力、Boyden小室分析方法检测细胞体外趋化能力及管腔形成法检测体外血管新生能力;通过real-time PCR及Western blot检测重组蛋白处理后的HUVECs KDR、FGFR-1表达。结果r SVMPI减少鸡胚尿囊膜新生血管密度指数,减弱由VEGF诱导的HUVECs趋化能力,抑制HUVECs体外新生小管的形成,降低HUVECs细胞KDR和FGFR-1的表达水平。结论r SVMPI可能通过阻断VEGF-KDR或b FGF-FGFR信号转导途径,发挥抑制血管生成的作用。Aim Toinvestigatetheeffectofrecombi-nant snake venom metalloproteinase inhibitor （rSVM-PI ） on neovascularization and its molecular mecha-nism.Methods Chickenchorioallantoicmembrane （CAM）assay was used to examine the antiangiogenic effect of rSVMPI.Alamar blue analysis was used to de-tect cell proliferation.Annexin V-FITC double labeling flow cytometry was used to assay cell apoptosis. Scratch marker was used to assay cell migration.Boy-den chamber analysis method was used to detect cells chemotaxis in vitro.Tube like structure（TLS）of HU-VECs was used to detect the ability of neovasculariza-tion in vitro.Real-time PCR and Western blot were used to assay the expressions of KDR and FGFR-1 inHUVECs. Results Thevasculardensityindex （VDI）of CAM was drastically decreased after rSVMPI treatment, chemotaxis of HUVECs in response of VEGF was inhibited in the presence of rSVMPI,TLS of HUVECs was less than control group.The expres-sions of KDR and FGFR-1 were down-regulated by re-al-timePCRandWesternblotassay.Conclusion rS-VMPI may inhibit neovascularization by blocking the VEGF-KDR or bFGF-FGFR signal transduction path-way.

关 键 词：蛇毒 基质金属蛋白酶抑制剂 重组蛋白 表达纯化 血管生成 信号转导 

分 类 号：R-332[医药卫生] R322.123