【作者】 杨亚培；

【导师】 刘贤锡； 董波；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 动脉粥样硬化(Atherosclerosis, AS)是一种严重威胁人类健康的常见病、多发病,也是冠心病、心肌梗塞及脑卒中的主要病因。目前研究认为AS是由多种致病因素引起的以内皮细胞损伤为基础、以血管的慢性炎症为特征的病理过程。目前研究认为动脉粥样硬化斑块分为稳定性斑块和不稳定性斑块两类。不稳定性斑块的突出特征是具有较大的脂质核、较薄的纤维帽和大量的炎性细胞浸润,胶原和平滑肌细胞数量少,是引起包括不稳定型心绞痛、急性心肌梗塞和心源性猝死在内的急性冠脉综合征(acute coronary syndrome, ACS)的病理基础。动脉粥样硬化斑块不稳定性与斑块的内部成分变化密切相关,斑块内的巨噬细胞、泡沫细胞、T-淋巴细胞等的多种炎症细胞通过分泌大量细胞因子及蛋白水解酶降解细胞外基质并促进平滑肌细胞凋亡,进而增加斑块易损性。肾素-血管紧张素系统(renin angiotensin system, RAS)由一系列酶、肽类、激素及其受体所组成,在AS发生发展过程中起重要作用。血管紧张素Ⅱ(AngⅡ)是RAS重要的末端活性产物,通过作用于1型受体(AT1)和2型受体(AT2)而发挥其生物学效应。肾素可特异性地切割血管紧张素原N-端第10与第11位氨基酸之间的肽键,催化血管紧张素原转化为血管紧张素Ⅰ,后者再转化为血管紧张素Ⅱ,从而激活RAS。这是RAS级联反应中的限速步骤。肾素是肾素原在激活酶催化作用下生成的一种蛋白酶。肾素作为Angll上游的RAS成员,在心脑血管遗传学研究中越来越受到人们的重视,其基因多态性成为高血压、冠心病和脑卒中的疾病的候选基因。近几年随着研究的深入,发现肾素和其前体肾素原,通过与肾素原受体结合,经由两种途径发挥生物学作用,一种是依赖血管紧张素途径,即催化血管紧张素原转化为AngI,进而在心血管系统中发挥重要作用；另一种是非血管紧张素途径,即通过不生成血管紧张素(Ang)的方式直接激活细胞内信号通路产生生物学作用。然而目前对于肾素/肾素原和其受体-肾素原受体的研究和认识还不全面深入,在对人肾素基因与疾病关系的研究中以与原发性高血压相关性研究较多,探讨其与动脉粥样硬化关系的研究报道很少。因此本研究首先构建了肾素基因过表达的腺相关病毒载体,于体内外实验中观察对动脉粥样硬化RAS系统及相关炎症因子、信号通路的影响,初步探讨肾素在动脉粥样硬化发生发展过程中的作用,为动脉粥样硬化的治疗提供新的理论依据和技术策略。越来越多的证据表明血脂代谢紊乱和肾素-血管紧张素系统(RAS)的激活在AS病理过程中起了重要作用。氧化型低密度脂蛋白(oxidized low density lipoprotein, OX-LDL)促进了内皮细胞和巨噬细胞表面的血管紧张素H1型受体(AT1R)的表达。植物血凝素样氧化低密度脂蛋白受体-1(Lectin-like oxidized low density lipoprotein receptor-1, LOX-1)在内皮细胞损伤、单核细胞与内皮细胞粘附及泡沫细胞形成过程中起了重要作用,是内皮细胞损伤的早期标志物。LOX-1在动脉粥样硬化中的作用机制尚不甚清楚,有研究报道,Ox-LDL与LOX-1的结合开始于NF-κB等转录因子的激活,同时增加细胞内ROS的形成。单核细胞与内皮细胞粘附并迁移到内皮下组织是AS早期的病理特征,单核细胞趋化因子(monocyte chemoattractant protein, MCP)-1作为一种重要的趋化因子在此病理过程中起重要作用。联合使用HMG-CoA还原酶抑制剂他汀类和AT1R拮抗剂(ARB)常用于治疗临床上伴有血脂代谢紊乱的高血压病人,并可以进一步减轻其AS程度,本文在观察了肾素过表达与AS关系后又观察并初步探讨了联合用药对AS炎症斑块及相关因子的影响。第一部分人肾素基因腺相关病毒表达载体的构建与鉴定目的构建人肾素基因腺相关病毒表达载体,并包装纯化出高滴度病毒,为研究肾素高表达对动脉粥样硬化影响的后续实验奠定基础。方法1.构建pDC316-Ren质粒；以原始质粒REN为模版设计引物扩增Ren片段,经SalI和BglII双酶切连接入载体质粒pDC316,转化得到pDC316-Ren质粒。酶切电泳和测序鉴定。2.以pSNAV2.0-tdRFP质粒构建pSNAV2.0-tdRFP-mCMV-Ren；以pDC316-Ren质粒为模版设计引物扩增mcmv-ren-polyA片段,经BamHI单酶切PCR产物和载体质粒PSNAV2.0-tdRFP,连接,转化得到pSNAV2.0-tdRFP-mCMV-Ren质粒。酶切电泳和测序鉴定。3. rAAV2-Ren-RFP的制备纯化,鉴定及滴度测定。将2.0-Ren-RFP质粒用Lipofectamine2000转染BHK-21细胞,经G418选择培养后,将此混合细胞株命名为BHK/HIF-2。将rAAV2-Ren-RFP的生产细胞株经HSV1-rc/△UL2感染(MOI为0.1)后,包装产生重组rAAV2-Ren-RFP, PCR扩增目的基因加以鉴定。鉴定正确的rAAV2-Ren-RFP用地高辛标记的CMV探针点杂交方法检测病毒液中rAAV2-Ren-RFP的物理滴度(以病毒基因组数/mL,vg/ml表示)。结果1.重组质粒pDC316-Ren经SalI和BglII双酶切预期产生大小分别3.8kb和1.2kb左右两条带,电泳结果与预期相符,该质粒经酶切鉴定正确,送测序正确。2.重组质粒pSNAV2.0-tdRFP-mCMV-Ren经BamHI单酶切预期产生大小分别为7.8kb和1.9kb左右两条带,而电泳结果与预期相符,该质粒经酶切鉴定正确,测序鉴定正确。3.重组rAAV2-Ren-RFP病毒经PCR鉴定,能够特异扩增出1.9kb左右大小的目的基因条带,证明该重组腺相关病毒携带有目的基因Ren。4.用地高辛标记的CMV探针点杂交方法检测病毒液中rAAV2-Ren-RFP的物理滴度(以病毒基因组数/mL,vg/ml表示)。通过计算Ren的拷贝数、再乘以与其杂交信号强度一致的病毒液样品的稀释倍数,得出rAAV2一Ren-RFP的物理滴度为8.3×1011vg/ml.结论1.成功构建重组质粒pDC316-Ren和pSNAV2.0-tdRFP-mCMV-Ren；2.成功构建重组rAAV2-Ren-RFP病毒载体；3.包装纯化出高滴度病毒,为后续研究工作奠定了基础。第二部分肾素基因过表达对动脉粥样硬化影响的体内外研究目的于体内外观察肾素过表达对动脉粥样硬化中RAS系统和相关炎症因子的影响,探讨肾素在动脉粥样硬化发展过程中的作用,为进一步进行动脉粥样硬化治疗的研究提供理论依据和技术策略。方法1.32只雄性新西兰大白兔先高脂喂养一周,然后行腹主动脉内膜损伤术,并继续高脂喂养12周形成AS斑块,于12周末随机分为3组即高脂组、rAAV2-RFP空载体组和rAAV2-Ren-RFP肾素基因表达组,于腹主动脉斑块明显处分别局部转染5×1010vg/只的rAAV2-RFP或rAAV2-Ren-RFP。16周末处死动物取材,HE和油红0染色分别观察AS斑块程度和血管内膜脂质情况；免疫组化检测AS斑块内肾素是否成功表达及各组巨噬细胞、VCAM-1的表达；RT-PCR检测各组VCAM-1、MCP-1mRNA的表达；Western blot检测各组间肾素表达水平。2.原代培养HUVECs,经传代,依据细胞形态及抗Ⅷ因子抗体鉴定后,取生长良好的3-8代用于实验。预先加入100M0IrAAV2-RFP孵育HUVECs,分别于24、48、72小时荧光显微镜下监测荧光强度,选取荧光最强时间点进行后续细胞实验—RT-PCR及Western blot检测,分析转染rAAV2-Ren-RFP后的renin及炎症因子VCAM-1、MCP-1等的表达,分析高表达的renin对AS相关炎症因子蛋白和mRNA表达的影响。Western blot检测高表达的肾素对相关信号通路蛋白表达的影响。结果1.HE染色显示肾素基因表达组血管内膜增厚,脂滴积聚；免疫组化显示肾素成功于转染斑块处表达,且肾素基因转染组的斑块内巨噬细胞浸润增多,炎症因子VCAM-1表达增加,斑块潜在不稳定性增加；RT-PCR检测AS相关炎症因子VCAM-1、MCP-1mRNA表达增加。Western blot显示肾素基因转染组肾素蛋白表达亦增加。2. rAAV2-RFP转染HUVECs不同时间发现,于48小时荧光最强,即选取48小时收获细胞进行后续检测。Western blot检测显示肾素基因转染组细胞的肾素蛋白大量表达,炎症因子蛋白表达量增多；RT-PCR检测各组细胞的mRNA表达水平,与蛋白表达趋势一致。Western blot检测信号通路蛋白表达显示p-ERK、p-P38MAPK、p-JNK表达增多。结论体内外实验显示,转染rAAV2-Ren-RFP组与rAAV2-RFP组相比,前者炎症因子的基因及蛋白表达均增加,表明肾素表达量增多时,AS相关炎症因子表达水平亦增加,斑块潜在不稳定性增加。调控RAS上游的肾素基因,或许可以为动脉粥样硬化的治疗提供新的技术策略。第三部分HMG-CoA还原酶抑制剂和ARB联合应用对动脉粥样硬化斑块及相关炎症因子的影响目的体内研究联合应用HMG-CoA还原酶抑制剂他汀类和ARB对AS斑块炎症及早期AS的影响,并进一步探讨其分子机制。方法将1.5-1.63kg重的雄性新西兰大白兔36只先行一周的正常饮食,然后被随机分为4组：高脂组(CH,n=9)即单纯饲以高脂饮食(1%胆固醇+5%猪油颗粒)；氯沙坦组(L,n=9)即高脂饮食+氯沙坦(25mg/kg/d);氟伐他汀组(F,n=9)即高脂饮食+氟伐他汀(lOmg/kg/d)；氟伐他汀+氯沙坦组(F+L,n=9)即高脂饮食+氟伐他汀(lOmg/kg/d)+氯沙坦(25mg/kg/d)。另取8只1.5-1.63kg重的雄性新西兰大白兔作为阴性对照组饲以正常饮食,以观察高脂饮食致AS斑块的病变程度。16周末收集兔耳缘静脉血检测血脂水平变化,剥离完整胸主动脉,HE染色观察AS斑块病变程度和内膜厚度；油红0染色检测AS斑块中脂质含量；免疫组化染色法检测并分析细胞因子MCP-1在斑块中含量以及巨噬细胞浸润程度和血管平滑肌细胞(SMCs)数量变化；RT-PCR检测各组MCP-1mRNA水平变化；Western blot检测各组p38MAPK蛋白含量变化。结果1.各组血脂水平变化：16周末兔耳缘静脉取血,检测发现氯沙坦组(L)与单纯高脂组(CH)相比,血总胆固醇和LDL-胆固醇水平无显著性差异,氟伐他汀组(F)与单纯高脂组(CH)相比,血总胆固醇和LDL-胆固醇水平明显降低(P<0.01),氟伐他汀+氯沙坦组(F+L)与单纯高脂组(CH)相比,血总胆固醇和LDL-胆固醇水平亦明显降低(P<0.01),提示氯沙坦单独使用并没有明显的降血脂作用。2.各组内膜厚度(I)和内膜厚度/中膜厚度(I/M)变化：与单纯高脂组(CH)相比,氟伐他汀组(F)和氯沙坦组(L)内膜厚度(I)和内膜厚度/中膜厚度(I/M)均明显降低(P<0.01),而氟伐他汀+氯沙坦组(F+L)的工和I/M进一步降低(分别与F、L组相比,P<0.01),提示两药联合使用对AS具有协同抑制效应。3.组织学检测HE染色发现,正常饮食组内膜结构正常,无斑块形成；CH组胸主动脉内膜明显增厚,管腔变窄,镜下可见大量泡沫细胞；F组和L组的内膜亦增厚,但与CH组相比,AS程度明显减轻；F+L组则进一步减轻了AS的程度。油红0染色,CH组内膜脂质含量丰富；F组和L组的脂质含量明显减少；F+L组脂质含量进一步减少。4.免疫组化：CH组增厚的内膜中可见大量巨噬细胞浸润,F组和L组均明显减少了巨噬细胞浸润(P<0.01),F+L组巨噬细胞浸润程度进一步减轻(P<0.01)；各组间SMCs表达无明显差异。免疫组化显示,CH组增厚的内膜中可见大量MCP-1阳性颗粒表达,与CH组相比,F组和L组的MCP-1阳性颗粒表达明显减少(P<0.01),而F+L组MCP-1阳性颗粒表达进一步减少(P<0.05)。5. RT-PCR:与CH组相比,F组和L组的MCP-1mRNA水平明显降低(P<0.01),F+L组的MCP-1mRNA水平进一步降低(P<0.05)。6. Western blot:与CH组相比,F组和L组的p38MAPKs表达水平明显下降(P<0.05),F+L组的p38MAPKs表达水平进一步降低(P<0.05)。结论1.氯沙坦单独使用并无显著降低AS血脂的作用；2. HMG-CoA还原酶抑制剂他汀类和ARB的联合应用,明显减轻了AS斑块内巨噬细胞浸润和内膜的厚度面积,减轻了AS程度并稳定了易损斑块。3.两者联合应用进一步降低了AS斑块内MCP-1及p38MAPKs的表达,对AS病变具有协同抑制作用,增加了斑块稳定性,为临床病人合理的联合用药提供进一步的实验依据。更多还原

【Abstract】 Atherosclerosis (AS) is a common disease which severely threatens human health. It is also a main reason of coronary artery disease and acute myocardial infarction. A variety of studies indicated that AS is a chronic inflammatory process based on the damage of vascular endothelial cells (VECs).Acute coronary syndromes, including unstable angina, acute myocardial infarction and sudden cardiac death, result from fissure, erosion, or rupture of a vulnerable atherosclerotic plaque. The intrinsic features that characterize a plaque vulnerable are an increased macrophage, T-lymphocyte, foam content, an increased lipid content, and reduced collagen and smooth muscle cell content. The characteristics of stable plaque are composed of solid fibrous or fibrocellular tissue and only small amount of extracellular lipid or no lipid and less inflammatory cell. The plaque nature depends on intrinsic features. Accumulation of macrophage, and T-lymphocyte in plaque may decrease its stability, because these inflammatory cells secrete cytokines and matrix metalloproteinases (MMP), which results in destruction of extracellular matrix and apoptosis of smooth muscle cell.Renin angiotensin system (RAS) has been demonstrated to play a critical role in the initiation and progression of atherosclerosis. AngiotensinⅡ(AngⅡ), a major substrate in RAS, stimulates atherosclerosis through various deleterious effects such as endothelial dysfunction, cellular proliferation and inflammation. AngⅡplays a main role in the RAS by interacting with its specific receptor, AngⅡtype 1 receptor (AT1R). AngiotensinⅠ(AI) and angiotensinⅡ(All) are generated from hepatic angiotensinogen (AGT), after a cascade of proteolytic cleavages involving a rate-limiting enzyme, renin. Renin is produced by the cleavage of prorenin, originating from the juxtaglomerular cells of the kidney. Renin and prorenin binding to the prorenin receptor not only target and facilitate angiotensin generation but also lead to activation of prorenin receptor signal transduction pathways, which is distinct from classical RAS signaling. In the study, we constructed the overexpression vector of human renin and detected the effect on atherosclerotic cytokines.A growing body of evidence has suggested that blood lipid metabolism disorders and angiotensinⅡ(AngⅡ) have synergistic effects on the occurrence of atherosclerosis, and the combination of dyslipidemia and the activation of the renin-angiotensin system (RAS) plays an important role in the pathogenesis of atherogenesis. Oxidized low-density lipoprotein (ox-LDL) can upregulate the expression of the angiotensinⅡtype 1 receptor (AT1R) on the surface of endothelial cells and macrophages. AngⅡfacilitates LDL oxidization and uptake by endothelial cells, smooth muscle cells and macrophages. The recruitment of monocytes into the arterial wall is one of the earliest events in the pathogenesis of AS. Recent studies showed that Lox-1 was a key molecule in the generation of endothedial dysfunction and played an important role in endothelial injury, monocyte adhesion to VECs and foam cells form in response to ox-LDL and angiotensinⅡ(AngⅡ). Monocyte chemoattractant protein-1 (MCP-1) is the main chemotactic factor and plays a crucial role in monocyte adhesion to endothelial cells. Monocyte and T-lymphocyte migrate into the subendothelial space by MCP-1, which plays a key role in the subendothelial recruitment of monocyte. The recruitment of monocytes into the arterial wall is one of the earliest events in the pathogenesis of AS. In the study, we investigated the effects and mechanisms of the combinational use of fluvastatin and losartan on the development of atherosclerosis and inflammation within the atherosclerotic plaques. Part One Construction and Identification of the Overexpression Vector of Human Renin GeneObjectiveTo construct and identification of the overexpression vector of human renin gene, purified and concentrated the recombinant overexpression virus.Methods1. The sequence encoding renin was amplified by RT-PCR and was cloned to SalⅠand BglⅡsites of pDC316, the positive plasmid constructs named pDC316-Ren. Then the pDC316-Ren plasmid was confirmed by electrophoresis and sequencing.2. The sequence mcmv-ren-polyA was amplified by PCR and was cloned to BamHI sites of pSNAV2.0-tdRFP, the positive plasmid constructs named pSNAV2.0-tdRFP-mCMV-Ren.Then the pSNAV2.0-tdRFP-mCMV-Ren plasmid was confirmed by electrophoresis and sequencing.3. pSNAV2.0-tdRFP-mCMV-Ren was transfected into BHK-21 by Lipofectamine 2000, then along with HSV1-rc/AUL2 into BHK/HIF-2 to package virus particles rAAV2-Ren-RFP. Then the rAAV2-Ren-RFP plasmid was confirmed by electrophoresis. The functional titer was determined.Results1. A fragment about 1.2 kb was amplified, the recombinant plasmid pDC316-Ren was constructed successfully and confirmed by SalⅠand BglⅡdigestion and DNA sequencing.2. A fragment about 1.9 kb was amplified, the recombinant plasmid pSNAV2.0-tdRFP-mCMV-Ren was constructed successfully and confirmed by BamH digestion and DNA sequencing.3. The recombinant virus rAAV2-Ren-RFP was packaged successfully and confirmed by DNA sequencing.4. The functional titer of the recombinant virus rAAV2-Ren-RFP is 8.3×1011vg/ml.Conclusions 1. The recombinant plasmids pDC316-Ren and pSNAV2.0-tdRFP-mCMV-Ren were constructed successfully2. The recombinant overexpression vector rAAV2-Ren-RFP was packaged successfully and concentrated effectively. It was necessary for subsequent research. Part Two The Effect of Human Renin Gene Overexpression on Atherosclerosis and Plaque Inflammation in vitro and in vivoObjectiveAssess the effect of human renin gene overexpression on atherosclerosis and plaque inflammation in vitro and in vivo, and provide a potential therapeutic target in cardiovascular disease.Methods1. Atherosclerosis (AS) was induced in the aortic arteries of New Zealand White Rabbits by endothelial injury after anesthesia and atherogenic diet for 12 weeks.2. Rabbits were randomly divided to 3 groups of high-cholesterol group (CH) and rAAV2-Ren-RFP group and rAAV2-RFP group (n=10 in each group) at the end of 12 weeks, the rAAV2-Ren-RFP group and rAAV2-RFP group were received a suspension of the recombinant renin overexpression virus (5×1010vg per rabbit) through a catheter into the abdominal aortic segments rich in atheromatic plaques and the rAAV2-RFP group were injected with empty vector.3. At the end of 16 weeks, the rabbits were anesthetized and their aortas were collected for histological and biochemical analysis. The protein and mRNA level of Renin, Macrophages, MCP-1 and VCAM-1 were detected by immunohistochemistry and RT-PCR.4. Human umbilical vein endothelial cells (HUVECs) were cultured and identified. HUVECs in passages 3 to 8 were utilized for in vitro experiments.5. HUVECs were incubated with 100MOI rAAV2-Ren-RFP, the rAAV2-RFP viral vector served as control group. Then HUVECs were harvested at 48 hours after gene transfection for Western blot and RT-PCR analysis. HUVECs were treated with rAAV2-Ren-RFP to evaluated the effect of renin expression on the mRNA and protein level of cytokines and signal transduction molecular.Results1. The protein and mRNA expression of renin gene were markedly increased at 48 hours, and the expression of VCAM-1 and MCP-1 were also increased at 48 2. Overexpression of renin leads to more macrophages and increased unstable lesion.ConclusionsRenin overexpression could increase the expression of VCAM-1 and MCP-1 and macrophage accumulation, leading to plaque unstability in the rabbit madel. It suggested that renin could play an important role in atherogenesis and maybe provide a potential therapeutic target in cardiovascular disease.Part Three The Effect of Combined Drugs on Atherosclerosis and Plaque InflammationObjectiveTo determine if the combination of fluvastatin and losartan has synergistic effects on relieving atherosclerosis and plaque inflammation induced by a high-fat diet in rabbits.MethodsThe rabbits (n=36) were provided with regular food for 1 week and were subsequently provided with high-cholesterol food (1% cholesterol and 5% pig oil particles) for 16 weeks to generate the atherosclerosis model. They were randomly divided into 4 groups. The high-cholesterol group (CH) contained rabbits (n=9) that were provided with high-cholesterol food without other treatments. The fluvastatin group (F) contained rabbits (n=9) that were provided with high-cholesterol food and fluvastatin (10mg/kg/d). The losartan group (L) contained rabbits (n=9) that were provided with high-cholesterol food and losartan (25mg/kg/d). The fluvastatin and losartan combination group (F+L) contained rabbits (n=9) that were provided with high-cholesterol food and treated with a combination of fluvastatin (10mg/kg/d) and losartan (25mg/kg/d). Blood samples were drawn from the marginal ear vein at the end of 16 weeks. Total cholesterol (TC) and LDL-cholesterol concentrations were evaluated. Macrophage infiltration, smooth muscle cells (SMCs) and MCP-1 protein expression were measured by immunohistochemistry. The protein and mRNA level of MCP-1 and p38MAPKs were analysis by western blot and RT-PCR. ResultsAlthough the combination of losartan and fluvastatin did not demonstrate a higher reduction in blood cholesterol levels than the individual losartan and fluvastatin groups, the combination of losartan and fluvastatin had synergistic effects on decreasing the intimal and media thickness of the thoracic aortas, which reduced the extent of macrophage infiltration and monocyte chemotactic protein-1(MCP-1) expression in the plaques.ConclusionsThe combinational use of losartan and fluvastatin significantly inhibited atherosclerotic evolution and reduced inflammation associated with atherosclerotic plaques.更多还原