【作者】 张丽芳；

【导师】 柯元南； 丁文惠；

【作者基本信息】 中国协和医科大学 ， 内科心脏病学， 2008， 博士

【摘要】 目的:1、研究人血浆UII浓度与冠状动脉病变程度的关系。2、通过大鼠胸主动脉球囊拉伤模型来观察损伤部位UII的表达及外源性应用UII对于损伤后重塑的影响,及能否通过UII拮抗剂Urantide拮抗内源性UII的作用。3、离体研究不同浓度UII对大鼠拉伤血管胶原的合成与分泌的影响。方法:1、在100名已知或怀疑冠心病患者中行冠脉造影检查,计算Micheal评分并测量血中UII浓度。2、采用大鼠胸主动脉球囊拉伤模型,假拉伤为对照,术后持续皮下渗透泵给予UII（1nmol/kg/h）或UII拮抗剂Urangtide（10nmol/kgh）21d,检测血管形态改变、UII及受体表达、胶原含量及分型、MMP-1蛋白和MMP-2/TIMP-2比。3、大鼠胸主动脉球囊拉伤术后7d,14d组行血管孵育,³H-Pro掺入研究不同浓度UII对胶原的合成与分泌的影响。结果:1、血浆UII浓度在冠脉正常或极轻微病变组（评分＜3）与冠脉病变严重组（评分≥9）间有显著差异（1.61±1.05ng/ml vs 2.50±1.62ng/ml,P=0.03）。血浆UII浓度与冠状动脉病变评分正相关,（r=0.213,P=0.034）,是继年龄、血糖异常、高血压之后的独立危险因素。2、与假拉伤组比,拉伤21d时损伤血管UII蛋白表达明显增加,UII受体GPR-14mRNA上调。予UII后损伤血管GPR-14mRNA进一步上调40%,增生内膜面积明显增加（（13±5）%vs（7±2）%,P＜0.05）,胶原含量增加,其中Ⅰ型胶原增加1倍,MMP-1蛋白的表达下降59.9%,MMP-2活性升高,TIMP-2蛋白减少。予Urantide后损伤血管GPR-14mRNA下调45%,增生内膜面积没有减少,胶原蛋白及mRNA水平、MMP-1蛋白均无明显改变,MMP-2活性升高,TIMP-2蛋白减少。3、UII以浓度依赖的方式刺激血管组织³H-Pro掺入。拉伤血管对UII的反应性增强,拉伤术后7d时UII10^-10、10^-9、10^-8mol/l分别使胶原合成增加87.7%、81.2%和56.8%,P＜0.05;胶原分泌增加34.6%、17.5%和17.2%,P＜0.05。拉伤14d时胶原合成及分泌的增加较7d减少。结论:1、在严重冠脉病变患者血浆中UII水平显著升高,并且UII水平与冠脉病变程度明显正相关。2、大鼠胸主动脉损伤后局部UII的表达增强,GPR-14mRNA水平上调,外源性应用UII后GPR-14mRNA水平进一步上调,促进新生内膜胶原沉积,加重损伤血管狭窄。10nmol/kg/h的Urantide能抑制GPR-14mRNA水平上调,但对胶原沉积无明显影响,未能表现出拮抗血管狭窄的作用,甚至对MMP-2/TIMP-2平衡有类UII的作用,其意义有待进一步研究。更多还原

【Abstract】 Objectives:1.To study the correlation between UrotensinⅡ（UⅡ） concentration and the severity of coronary artery disease（CAD）.2.To evaluate the exogenous UII’s effection on injured vessel,especially the extracellular matrix response.In addition the effect of Urantide,a selective peptidic UT receptor antagonist,was assessed too.3.To evaluate the effection of UrotensinⅡon collagen synthesis and secretion in vitro.Methods:1.We studied UII concentrations in 100 patients with known or suspected CAD referred for coronary arteriography.Micheal score system was used to estimate the severity of CAD.2.Stenosis model of thoracic aorta 21 days after balloon injury was established in male Wistar rats,which were divided into 4 groups（n=5）,including sham injured,injured,UII（1nmol/kg/h,infused by osmotic mini-pump） and Urantide（10nmol/kg/h） group.H-E staining and Masson-Trichrome staining were used for morphometric analysis,RT-PCR for the mRNA expressions and immunohistochemical staining assay for protein expression.3.Thoracic aortas were separated 7days and 14 days after balloon injury（4 rats per group） and treated with UrotensinⅡ,collagen synthesis and secretion were measured by ³H-proline incorporation.Results:1.UII concentration was higher in severer group（score≥9） than in normal or nearly normal group（score＜3）,（2.50±1.62ng/ml vs 1.61±1.05ng/ml,P=0.03） UII concentration correlated with CAD severity（r=0.213,P=0.034）.By multiple regression analysis,UII is one determinant of the severity of CAD,other than age, abnormal glucose,hypertension and gender.2.①Compared with sham injured vessel,UII was increased significantly in injured vessel,and GRP-14mRNA was upregulated.②In UII group GRP-14mRNA further upregulated,the intimal hyperplasia was markedly increased（（13±5）%vs （7±2）%,P＜0.05）,collagen content was also increased,typeⅠcollagen increased 1 fold,typeⅢcollagen decreased 64%,while no significant difference in mRNA levels was found.MMP-1 decreased significantly（3.33±0.82 vs 8.30±1.81,P＜0.05）. MMP-2 activity increased 1.25 fold,TIMP-2 protein decreased by 73.3%.③In Urantide group,compared with injured group,GRP-14mRNA dowuregulated,the intimal hyperplasia didn’t reduce（（9±3）%vs（7±2）%,P＞0.05）,collagen protein and mRNA had no difference,MMP-1 protein had no significant difference,the activity of MMP-2 increased 1.29 fold,TIMP-2 protein decreased by 55.4%.3.UII stimulated the collagen synthesis in non-injured vessel in a concentration-dependent manner.In aorta 7days after balloon injury,the collagen synthesis increased by 87.7%,81.2%,56.8%（all P＜0.05） than in non-injured vessel stimulated by 10^-10,10^-9 and 10^-8mol/l UII respectively,and collagen secretion increased by 34.6%,17.5%and 17.2%（all P＜0.05）.The synthesis and secretion increase 14 days later is less than that of 7days later.Conclusions:1.UII is elevated in severe CAD and there is a significant relationship between UII concentration and CAD severity.2.In injured vessel,UII expression increases,and GRP-14 mRNA upregulates.Exogenous UII stimulates GRP-14 mRNA upregulation,collagen accumulation,and increases MP-2/TIMP-2 rate which may prolong vascular remodeling contributing to intimal hyperplasia.Urantide（10nmol/kg/h） can antagonize GRP-14mRNA upregulation,but has no effect on collagen accumulation and has a similar tendency on MMP-2/TIMP-2 balance,so in present study,Urantide has no protective effect on blood vessel stenosis.更多还原