【作者】 万静；

【导师】 马业新；

【作者基本信息】 华中科技大学 ， 心内科， 2006， 博士

【摘要】 研究背景过氧化物酶体增殖物激活受体（PPARs）是核受体超家族成员，有PPARα、PPARγ和PPARβ／δ3种亚型，这三种亚型由不同的基因编码，但它们的蛋白质结构十分相似。PPARs与配体结合后，与视黄酸类受体形成二聚体，然后与所调节基因的启动子上游的过氧化物酶体增殖物反应元件（即PPRE，是一段DNA序列，它与增殖物结合后可以调节一些基因的表达）结合而发挥转录调控作用。过氧化物酶体增殖物激活受体γ（PPARγ）是PPARs中最具脂肪组织特异性的成员，它能够调节脂质代谢、脂肪细胞分化和胰岛素敏感性。PPARγ基因分为PPARγ1、PPARγ2、PPARγ3，位于第3号染色体2带5区位点上，由6个外显子编码，三种亚型有共同的外显子，不同之处在它们的5’端，各自具有自身的启动子。虽然不同的PPARγ组织分布均很广，但它们都表达在大肠、脂肪组织中，近期研究表明，它尚在单核／巨噬细胞、泡沫细胞、心脏血管平滑肌细胞、内皮细胞、动脉粥样硬化、肿瘤病灶中表达。胰岛素增敏剂噻唑烷二酮类药物是其高效配体。动脉粥样硬化是一种多因素疾病，可以引起心脏、脑等重要脏器缺血甚至梗死。动脉粥样硬化斑块病变的形成包括单核／巨噬细胞、T细胞的聚集，泡沫细胞的形成、平滑肌细胞的迁移和增殖，以及细胞碎片堆积、纤维帽的形成。最近研究发现PPARγ在动脉粥样硬化损伤处和泡沫细胞中都有较高的表达，并与肥胖相关的代谢性疾病，如高脂血症、胰岛素抵抗和冠心病等发病有关。研究目的本文拟从基因水平研究PPARγ与动脉粥样硬化的关系。首先从人群的基因多态性研究入手，了解该基因C161→T基因多态性与冠状动脉粥样硬化性心脏病患者冠脉病变程度和糖脂代谢的关系；然后建立心梗的动物模型，观察PPARγ激动剂马来酸罗格列酮对心肌梗死大鼠血流动力学、梗死面积、心肌细胞凋亡率的影响，研究组织PPARγ基因表达对大鼠心肌梗死的作用；再从细胞水平给予罗格列酮干预，观察其对PPARγ与ET-1表达水平的影响，了解两个基因之间的相关性，同时观察炎症因子TNF-α与PPARγ的关系，了解PPARγ对内皮功能有无保护作用以及对炎症反应的作用；最后选择siRNA方法减少PPARγ的表达量，观察ET-1的变化，从另一方面了解二者之间的关系。研究方法1、运用聚合酶链式反应及限制酶片段长度多态性技术分析292例研究对象（包括89例正常健康人，203例冠心病患者）中PPARγC161→T基因多态性；结合放射免疫技术检测血浆胰岛素水平，HOMA方法评价胰岛素抵抗状况，冠状动脉造影检查冠状动脉病变，采用Gensini积分评估冠脉病变的程度，以及临床常规检测项目：空腹、餐后2小时血糖、血脂、身高、体重等指标，综合分析基因频率、等位基因频率分布及不同基因型与临床资料、生化指标的关系，并对该基因突变的危险性进行评价。2、结扎大鼠左冠状动脉主干制作心肌梗死模型，分为非罗格列酮治疗组（AMIA）和罗格列酮(5mg.kg^-1／天)治疗组（AMIB），以假手术（Sham）组作为对照组，手术过程中仅穿线绕过冠状动脉而不结扎。通过MP-150生理记录仪检测血流动力学变化，HE染色检测梗死面积，流式细胞术检测心肌细胞凋亡率，RT-PCR检测心肌组织PPARγ基因表达，观察罗格列酮对上述指标的影响。3、采用罗格列酮干预体外培养的人正常脐静脉内皮细胞以及AngⅡ诱导的脐静脉内皮细胞24小时，通过RT-PCR、适时定量PCR（quantitative real-time PCR）检测PPARγ及ET-1的mRNA表达量，采用Western blot法检测PPARγ及ET-1前体的蛋白质表达水平。同时用ELISA法检测内皮细胞TNF-α的分泌量。4、采用siRNA Expression Cassette方法干预体外培养的人正常脐静脉内皮，通过RT-PCR、real-time PCR及Western blot法检测PPARγ及ET-1的mRNA、蛋白质表达水平。结果1、PPARγC161-T基因多态性与冠状动脉粥样硬化性心脏病患者冠脉病变程度和糖脂代谢的关系（1）正常组T等位基因频率为0.213，C等位基因频率为0.787；冠心病组T等位基因频率为0.192，C等位基因频率为0.808；两组间基因型频率及C、T等位基因频率分布无显著性差异（P＞0.05）。存在冠状动脉病变的冠心病组以CC基因型为主，与T等位基因携带者（CT+TT）相比较差异有显著性（P＜0.05），且T等位基因携带者（OR:0.56，95％CI:0.24-0.63）冠心病危险性低于CC基因型（OR:1.92，95％CI:1.09-2.54）。（2）142例做过冠脉造影的冠心病患者的冠脉病变程度经Gensini积分评估后，发现CC型评分明显高于CT及TT型（P＜0.05）。（3）冠心病患者中CC基因型的apoB明显高于T等位基因携带者（CT+TT）（1.02±0.22与0.94±0.23，P＜0.05），胰岛素抵抗指数差异无显著性（P＞0.05）。2、PPARγ激动剂罗格列酮对心梗大鼠心脏血流动力学、组织变化、心肌细胞调亡及PPARγ基因表达的影响（1）大鼠心梗之后心功能下降，AMIA组与假手术组比较MAP、LVSP和±dp／dt均明显减少，分别为105.60±10.71与124.17±7.18，130.63±7.24与150.34±6.82，3805.30±244.86与5940.83±400.77，2749.00±131.45与4634.75±333.96，LVEDP增高(（15.5±2.35与4.52±0.57，P＜0.05）。（2）罗格列酮治疗14天后，与AMIA组比较LVEDP下降（10.14±2.28与15.5±2.35），心梗面积减少33％，病理组织学改变较AMIA组明显减轻。（3）大鼠心肌梗死后心肌细胞凋亡率较假手术组升高21.15倍，经罗格列酮治疗14日后，心肌细胞凋亡率较AMIA组明显降低（16.04±2.26与26.44±3.51，p＜0.01）。（4）经罗格列酮治疗后，心肌梗死大鼠心肌组织PPARγ基因表达量较AMIA及假手术组明显增加（2.352±0.159，1.574±0.196与0.491±0.078，p＜0.001）。3、罗格列酮干预体外培养的人正常脐静脉内皮细胞以及AngⅡ诱导的脐静脉内皮细胞后对PPARγ、ET-1基因表达及TNF-α分泌的影响。（1）罗格列酮干预正常细胞后使PPARγmRNA和蛋白质水平表达量上调，ET-1mRNA及ET-1前体蛋白表达量减少（p＜0.001），mRNA及蛋白表达量的变化之间均有一定的相关性（r=0.914，p=0.011；r=0.999，p=0.028）。（2）AngⅡ刺激脐静脉内皮细胞后PPARγmRNA及蛋白表达量上调，ET-1mRNA及ET-1前体蛋白表达量也上调；加入罗格列酮24小时后PPARγ表达量明显上调，而ET-1表达量明显下调（p＜0.05）。（3）AngⅡ刺激内皮细胞后TNF-α分泌量呈浓度依赖性增高（p＜0.05）；加入罗格列酮后TNF-α分泌量明显减少（p＜0.05）。4、针对PPARγ基因设计的siRNA Expression Cassette 1（1326）使PPARγmRNA及蛋白表达量下调，ET-1mRNA表达量增多，二者之间mRNA表达量的灰度比值变化有相关性（r=0.995，p=0.042），siRNA Expression Cassette 2、3、4片段未能发挥抑制效应。结论1、在中国汉族冠心病及正常人群中PPARγC161→T基因型频率分布及等位基因频率分布无显著性差异；做过冠脉造影的冠心病患者中，CC基因型患者Gensini积分明显高于CT+TT型，提示C等位基因可能与冠脉疾病严重程度相关联；PPARγC161→T与冠心病有重要的相关性，T等位基因携带者可以减少冠心病的危险性。2、罗格列酮能有效降低大鼠心肌梗死后心肌细胞凋亡，减少心肌梗死面积，这可能与其增加梗死后心肌组织中PPARγ基因表达量，发挥了PPARγ对心肌细胞及局部血管的保护作用有关。3、罗格列酮可以通过增加PPARγ的表达量在转录水平和翻译水平抑制ET-1及其前体在人脐静脉内皮细胞的基因表达，还可以抑制人脐静脉内皮细胞炎症因子TNF-α的分泌。4、干扰RNA片段siRNA Expression Cassette 1（1326）对PPARγ的基因表达有抑制作用，可以在转录水平抑制PPARγ在人脐静脉内皮细胞的基因表达，同时使ET-1基因表达上调。更多还原

【Abstract】 ObjectivesThe nuclear receptor superfamily of PPARs consists of isoform α, γ, and β/δ. Although these different members are encoded by separate genes, they have a similar protein structures. PPARs regulate gene expression by binding as heterodimers with retinoid X receptors （RXRs） to specific PPAR response elements （PPRE） in the promoter regions of specific target genes. PPARγ is found predominantly in adipose tissue, where it plays a crucial role in adipocyte differentiation, lipid metabolism and insuline resistance. The PPARγ gene, located at 3p25-24, gives rise to three distinct mRNAs, i.e. PPARγ1, PPARγ2 and PPARγ3. All three PPARγ subtypes contain the common exons 1-6, each differs in their 5’ ends and each is under control of their own promoter. PPARγ is mainly expressed in the intestine and in the adipose tissue. Furthermore, PPARγ is expressed in vascular cells including endothelial cells, smooth muscle cells, monocyte/macrophage cells and foam cells. It can be detected out in atherosclerosis and tumor tissue. The antidiabetic glitazones are high-affinity agonists of PPARγ.Atherosclerosis is a multifactorial disease which may result in ischemia of the heart and brain, and infarction. The formation of atherosclerotic lesions involves attraction of monocytes/macrophages and T lymphocytes. Intermediate and advanced lesions typically consist of lipid-laden monocytes/ macrophages （foam cells）, migrating and proliferating smooth muscle cells, and the accumulation of cell debris and/or the presence of fibrous caps. Recent studies showed that PPARγ expressed in atherosclerotic lesions and macrophage foam cells, and was associated with obesity-related metabolic diseases such as hyperlipidemia, insulin resistance, and coronary artery disease （CAD）.Our objective in this study was to explore the relationship between PPARγ and ET-1 in gene level. At first, we investigated the PPARγC161→T substitution in our well-characterized hospital- based patients of coronary artery disease, in order to observe the relation between PPARγ gene C161→T polymorphisms and the severity of coronary lesion, and the relationship of the polymorphisms with lipid and glucose metabolism in Han Race Chinese. Secondly we established a model of myocardial infarction in rat to observe the effect of PPARγ agonist Rosiglitazone maleate on the ventricular haemodynamics, the histological change, the apoptosis level of cardiomyocytes and PPARγ gene expression after myocardial infarction in rats. Thirdly, we studied the effects of Rosiglitazone on the expression of PPARγ and endothelin-1 （ET-1）. We wanted to understand the relationship between the two genes, observe the relation between PPARγ and inflammation elements, such as Tumor Necrosis Factor-α （TNF-α）, and understand the effects of PPARγ in protecting the function of endothelial cell and reducing the inflammation response. At last, we observed the expression of ET-1 under the condition that the expression of PPARγ was reduced with the siRNA technique, and by this way we reconfirmed the correlation of the two genes from another way.Methods1、 292 subjects were investigated in this study, including 89 healthy persons, 203 cases diagnosed as CAD. PPARγC161→T gene polymorphism was determined by polymerase chain reaction and restriction fragment length polymorphisms, the blood glucose and the blood lipoprotein were detected, and body height and body weight were measured. The coronary artery lesions were detected by coronary angiography and analysed quantitatively by Gensini score method. The risk factors of CAD were estimated, and the frequencies of PPARγC161→T genotypes and the "T" allele in the CAD and healthy groups were observed.2、Acute myocardial infarction （AMI） model of rat was established by ligation of the left coronary artery. AMI rats were divided into two groups: AMIA group （AMI rats without any treatment） and AMIB group (AMI rats were treated with PPARγ agonist thiazolidinedione - Rosiglitazone 5mg.kg^-1/day). The rats in sham group underwent the same procedures but without tying the LAD artery. The infarct size and the changes of the cardiac structure after infarction were assessed by the method of hematoxylin-eosin stain. The cardiac function was evaluated by the physiological signal recording system. The apoptosis level was examined with the method of flow cytometry. The expression of PPARγ was examined by RT-PCR.3、 At 24 hour after administrated of Rosiglitazone, the mRNA of PPARγ and ET-1 were determined by RT-PCR and quantitative real-time PCR, the protein of PPARγ and ET-1 precursor were detected by western-blot in the normal and the Angiotensin II （AngII） induced human umbilic vein endothelial cell （ HUVEC） in vitro. The secretion of TNF-α in HUVEC was measured with ELISA.4、The HUVEC was intervened with siRNA Expression Cassette segments in vitro, 24 hours later, the mRNA of PPARγ and ET-1 were detected with RT-PCR, quantitative real-time PCR, the protein of PPARγ was measured with western-blot.Results1、Relationship of PPARγC161-T gene polymorphism and coronary artery disease（1） In normal healthy group, "T" allele frequency was 0.213, "C" allele frequency was 0.787, and in CAD group "T" allele frequency was 0.192, "C" allele frequency was 0.808. There was no significant difference between the two groups.（2） There was a significant association between PPARγC161→T genotypes and the number of significantly disease vessels. "T" allele carriers were far more frequent in patients without than those with significantly diseased vessels （P<0.05）, and the CAD risk in the "T" allele carries （OR: 0.56, 95%CI: 0.24-0.63） was much lower than that in the CC homozygote （OR: 1.92, 95%CI:1.09 -2.54）. The results showed that Gensini score in patients with CC genotype were markedly higher than that in patients with ’T’allele （p<0.05）.（3） apoB was obviously higher in patients with CC homozygote than those with "T" allele carriers （1.02±0.22 与 0.94±0.23,P<0.05）, and there was no different in HOMA IR.2、 The influences of Rosiglitazone on the ventricular haemodynamics, the histological change, the apoptosis level of cardiomyocytes, and PPARγ gene expression in Rats’myocardial infarction models（1） The cardiac function declined after AMI. AMIA group compared with the sham group, MAP, LVPSP and ±dp/dt_max declined （105.60±10.71 VS 124.17±7.18, 130.63±7.24 VS 150.34±6.82, 3805.30±244.86 VS 5940.83±400.77, -2749.00±131.45 VS ⁴634.75± 333.96,p<0.05, respectively）, but LVEDP rised significantly （15.5±2.35 VS 4.52±0.57,P＜0.05）.（2） After 14 days treatmented by Rosiglitazone, the LVEDP declined （10.14±2.28 VS 15.5±2.35, p<0.05）, and infarct size decreased 33% compared with AMIA group. The change of the cardiac structure after infarction in AMIB group was better than in AMIA group.（3） AMIA rats’ apoptosis level was as high as 21.15-fold of the sham group. Compared with AMIA group, after 14 days treatment by Rosiglitazone, the apoptosis level was decreased significantly （16.04±2.26 VS 26.44±3.51,p<0.05）.（4） Compared with AMIA group and the sham group, expression of PPARγ was increased obviously in AMIB group （2.352±0.159 VS 1.574±0.196 VS 0.491±0.078, p＜0.001）.3、 The effect of Rosiglitazone on gene expression of the nomal and Ang II - induced human umbilia vein endothelial cell in vitro（1） The mRNA and protein expression levels of PPARγ were increased significantly by Rosiglitazone, on the contrary, the level of ET-1 mRNA and its precursor pretein expression reduced （p<0.001）. There was relationship between two genes expression （ r=o.914, p=0.011; r=0.999, p=0.028）.（2） The mRNA expression of PPARγ and ET-1, and the protein expression of PPAR-γ and ET-1 precursor were markedly increased in HUVEC stimulated by Ang II as compared with that in the control group. 24 hours after administration of Rosiglitazone, the mRNA expression and protein expression of PPARγ rised, at the same time the expression of ET-1 reduced （P<0.05）.（3） The secretion of TNF-α increased by Ang II in a dose- dependent manner; and restrained by Rosiglitazone （p<0.05）.4、siRNA Expression Cassette 1（1326） down-regulated the mRNA and protein espression of PPARγ and up-regulated ET-1 gene mRNA expression at the same time. A link was found between two genes（r=0.995, p=0.042）.Conclusion1、 In the Han race Chinese, the distributing trend of PPARγC161→T gene polymorphism in the healthy group is as the same as that in the patients with CAD group; The Gensini score of the coronary artery angiography in persons with CC genotype are significantly higher than that in the persons with "T" allele. It means that there is a lower risk of CAD in the patients with "T" allele.2、Rosiglitazone can decrease infarct size and apoptosis level of AMI in rats. The effect of Rosiglitazone may be associated with the increase of PPARγ gene expression, which can protect the function of cardiac muscle cell and vascularity.3、 The expression of PPARγ in the mRNA and protein level can be enhanced, the ET-1 gene expression in the mRNA level and ET-1 precursor expression in the protein level, and TNF-α secreted from the vascular wall can be suppressed by Rosiglitazone through the way of increased PPARγ gene expression in HUVEC.4、siRNA Expression Cassette 1（1326） can cause an inhibition of PPARγ expression which lead to an increase of ET-1 expression. Therefore, both of PPARγ and ET-1 play a key role in formation of the Atherosclorsis.更多还原