细胞因子-基质金属蛋白酶轴促进小鼠血管内膜增生的机制研究  

Role of cytokine-matrix metalloproteinase axis on promoting vascular neointima hyperplasia in mice

作　　者：刘音[1] 宁文虎[1] 申星花[1] 郭殿龙[1] 郭玲[1] 

机构地区：[1]哈尔滨医科大学附属第四医院心血管内科,150001

出　　处：《中华心血管病杂志》2016年第11期961-967,共7页Chinese Journal of Cardiology

摘　　要：目的 观察肿瘤坏死因子-α（TNF-α）和血小板源性生长因子（PDGF）在基质金属蛋白酶（MMP）9和（或）MMP2基因敲除小鼠中促进血管内膜增生的情况,并探讨其作用机制.方法 将C57BL/6N小鼠按随机区组法分为对照组、MMP9基因敲除（MMP9-/-）组、MMP2基因敲除（MMP2-/-）组和MMP9/2基因敲除（MMP9/2-/-）组,每组10只小鼠.取对照组小鼠2只,将其股动脉线性损伤,逆转录酶-聚合酶链反应（RT-PCR）检测TNF-o和PDGF-bb的mRNA表达水平,Western blot法检测MMP9和MMP2的蛋白表达水平,用Odssey图像分析软件测得的蛋白条带灰度值;另取2只对照组小鼠,一只在其血管内膜损伤周围注射外源性TNF-α （5 ng/ml,0.10 ml）、TNF-α（0.05 ml）＋ MMP抑制剂SB-3CT （0.50 ng/ml,0.05 ml）,另一只注射PDGF-bb（10 ng/ml,0.10 ml）、PDGF-bb（0.05 ml）＋ SB-3CT（0.05 ml）,观察损伤后2和4周小鼠血管内膜增生情况.MMP9/2-/-组小鼠股动脉内膜线性损伤后2和4周观察其内膜增生情况.MMP2-/-组小鼠股动脉内膜损伤后,在其损伤周围注射TNF-α0.1 ml,MMP9-/-组小鼠损伤血管内膜周围则注射PDGF-bb 0.1 ml,观察各组小鼠血管内膜增生情况.采用Elastica-van Gieson染色法观察小鼠损伤后股动脉内膜增生情况,SigmaPlot测量内膜和中膜面积,并计算其比值.最后,取MMP9-/-组和MMP2-/-组小鼠股动脉血管平滑肌细胞（VSMC）培养基,分别给予TNF-α和PDGF-bb干预,进行迁移和增殖实验,测定迁移和增殖细胞数.结果 （1）血管内膜损伤后TNF-α、PDGF、MMP9和MMP2于不同阶段被诱导：对照组小鼠TNF-α、PDGF-bb mRNA表达在血管内膜损伤后1d达高峰,MMP9和MMP2的蛋白表达水平在血管内膜损伤后7和28 d表达水平较高.（2）TNF-α、PDGF-bb分别于血管内膜损伤后不同阶段诱导内膜增生：血管内膜损伤2周时,TNF-α干预较未干预的对照组小鼠血管内膜增生更为明显,内膜比值分别为2.21 ± 0.05和1.55±0.03（P =0.Objective To observe the effects of tumor necrosis factor-or （TNF-α） and platelet derived growth factor （PDGF） on vascular neointimal hyperplasia on matrix metalloproteinase 9/2 gene knockout （MMP9/2-/-） mice and explore related mechanisms.Methods Mice of control group,MMP9-/-group,MMP2-/-group and MMP9/2-/-group were studied.Femoral artery was injured by transluminal wire,the mRNA expression levels of TNF-o and PDGF on femoral artery were detected by RT-PCR;the protein expression of MMP9 and MMP2 were assessed by Western blot on day 0,1,3,7,14and 28 post injury.Mice in control group received TNF-α（5 ng/ml,0.10 ml）,TNF-α（0.05 ml） ＋ MMP inhibitor SB-3CT （0.50 ng/ml,0.05 ml） injection,or PDGF-bb （10 ng/ml,0.10 ml） and PDGF-bb （0.05 ml） ＋ SB-3CT（0.05 ml） injection around injured artery,intimal hyperplasia at 2 and 4 weeks after injury was observed.Intimal hyperplasia at 2 and 4 weeks after injury was also observed in MMP9/2-/-mice.TNF-α（5 ng/ml,0.10 ml） was injected to MMP2-/-mice,PDGF-bb （0.1 ml） was injected to MMP9-/-mice around injured artery,intimal hyperplasia at 2 and 4 weeks after injury was observed.The degree of neointimal hyperplasia were observed by the Elastica-van Gieson staining and the area of neointima and media of the arteries were measured by SigmaPlot and intima ratio was calculated.Vascular smooth muscle cell （VSMC） mediums of MMP9-/-and MMP2-/-mice were stimulated by TNF-α and PDGF-bb,respectively,and migration assay,and proliferation assay were performed,relative migration and proliferation cells numbers were counted.Results （1） mRNA expression of TNF-o （235.33 ± 23.68） and PDGF-bb （3.30 ±0.56） in femoral arteries peaked at 1 day after injury,while MMP9 or MMP2 protein expression peaked at 7 or 28 days after injury.（2） In control mice,TNF-α intervention significantly enhanced intimal hyperplasia at 2 weeks after injury （2.21 ±0.05 vs.1.55 ±0.03 in blank control group,P 〈 0.05）,while PDGF-bb intervention signifi

关 键 词：血管 肿瘤坏死因子-Α 血小板源性生长因子 基质金属蛋白酶类 内膜增生 

分 类 号：R543[医药卫生—心血管疾病]