【作者】 王云英；

【导师】 包振民； 胡景杰；

【作者基本信息】 中国海洋大学 ， 遗传学， 2007， 博士

【摘要】 高血压是一种复杂的临床疾病,发病机理非常复杂,涉及与动脉粥样硬化和血栓形成有关的多个基因和环境因素以及它们之间的相互作用。通常,富含脂质的动脉粥样硬化斑块的破裂是急性血栓形成的基础,是从动脉硬化性疾病的稳定期或亚临床期到急性心肌梗死、缺血发作和外周血栓形成的转变。迄今,对这些动脉血栓性疾病的预防很大程度上限于环境因素,但是有一半的血栓栓塞查不到这些危险因素,流行病学研究表明,环境因素不能完全解释血栓的形成。遗传因素在冠心病的发生发展过程中起重要作用。因此,当前的研究焦点已经从外界环境因素转移到了血栓形成和动脉粥样硬化的分子遗传学基础上,以进一步了解动脉血栓形成的病理机制。越来越多的临床和实验证据表明,原发性高血压(Essential hypertension, EH)存在促凝高凝状态。先前的研究已经发现几个基因突变是促凝的危险因子,其中血栓调节蛋白(thrombomodulin,TM)基因、转化生长因子-β1(transforming growth factor-beta-1,TGF-β1)基因和白细胞介素-10(interleukin 10, IL-10)基因是非常重要的有潜力的候选基因。本研究旨在分别评价这三个基因多态性对高血压及其心血管并发症发生发展的影响。1、可溶性血栓调节蛋白基因可溶性血栓调节蛋白(Soluble thrombomodulin ,sTM)作为内皮损伤或内皮功能障碍的特殊血浆标志物可能与高血压并发症有关,并可能影响血压高低。我们选择高血压患者286例,高血压伴冠心病(coronary heart disease,CHD)患者113例和年龄性别相匹配的健康对照者213例,采用聚合酶链反应-限制性片段长度多态性(PCR–RFLP)分析方法检测TM -33G>A多态性,流式细胞分析技术检测TM在单核细胞表面的表达以及ELISA测定血浆sTM水平。结果单核细胞表面的TM水平在高血压患者尤其是高血压伴CVD患者中呈现低表达,并且与TM–33位点多态性相关。但sTM和TM–33位点A等位基因频率在高血压组和健康对照组之间以及在高血压组和CVD组之间无显著性差异。同样基因型分布在各组之间亦无显著性差异,GG基因型与GA+AA基因型之比在EH组为77%:23%;CVD组为79.7%:20.3%;对照组为81.2%:18.8%。(与对照组比较均P>0.05)。用高血压公认的危险因素(吸烟,饮酒,胆固醇)校正后,TM–33位点基因型分布和等位基因频率仍无显著性差异。结论TM -33G>A多态性似乎与汉族人群高血压及CHD发病无明显相关。TM基因多态性可能影响其在细胞表面的表达,细胞表面TM低表达可能是高血压CHD发病的危险因素,但这种表达差异可能在高血压发生发展过程中被其他因素的影响所掩盖。2、转化生长因子-β1基因流行病学、临床和试验研究提示转化生长因子-β1(transforming growth factor-beta-1,TGF-β1)可能在高血压的发病过程中起重要作用,TGF-β1是一种多潜能的生长因子,能够调节细胞的生长和分化,调节细胞外基质及其修复,从而进一步调节炎症和免疫应答,参与血管的修复,而这一过程与高血压血管和靶器官损害的发病有关。TGF-β1也可以通过刺激内皮素-1和/或肾上腺素的分泌调节血压。本研究旨在探讨循环水平的TGF-β1蛋白与血压的相关关系,以及TGF-β1基因多态性各等位基因及基因型在高血压病冠心病患者中的分布频率,分析其基因型和血清水平与高血压病冠心病的相关关系。方法采用序列特异性引物聚合酶链反应( PCR-SSP),检测286例高血压病患者、113例高血压冠心病患者及213名正常对照者TGF-β1基因+869T/C、+915G/C多态性,同时采用双抗体夹心ABC-ELISA法测定血清TGF-β1水平。结果高血压冠心病组血清TGF-β1水平显著高于高血压组和对照组,高血压组显著高于对照组,均P<0.05。TGF-β1基因+915G/C多态性在高血压组、冠心病组和正常人群中的分布差异均无统计学意义(均P>0.05) ,而TGF-β1+869T/C基因多态性在冠心病组和正常人群中的分布差异存在统计学意义(均P<0.05),等位基因频率的相对风险分析发现, C等位基因携带者患高血压病冠心病的风险是T等位基因的1.681倍(OR =1.681, 95% CI: 1.213～2.328),提示TGF-β1+869位点C等位基因可能是冠心病的危险因素之一。携带C等位基因的高血压冠心病患者血清TGF-β1水平显著高于不携带者,呈现CC基因型者TGF-β1水平显著高于TC基因型者,TC基因型者TGF-β1水平显著高于TT基因型者(CC:TC:TT为7.06±0.69: 6.84±0.75: 6.12±0.58。表明TGF-β1 +869T/C位点的等位基因仅有一个发生改变即可以影响其蛋白水平的表达,C等位基因是TGF-β1的高表达基因。结论TGF-β1基因+ 869T/C多态性与高血压、冠心病的发病具有相关性,其中C等位基因可能是高血压、冠心病发病的遗传易感基因;携带C等位基因的个体可能通过促进TGF-β1的高度表达进而增加了高血压冠心病的发病风险,并且TGF-β1对冠心病的影响可能独立于血压水平之上,支持TGF-β1对冠心病的影响可能是通过细胞外基质的沉积而发挥作用的理论。而TGF-β1基因+915G/C多态性与高血压和冠心病的发病风险无显著关联。3、白细胞介素-10基因白介素-10(interleukin-10, IL-10)是由巨噬细胞、T细胞和B细胞产生的细胞因子,通过抑制促炎性细胞因子和B细胞的活性发挥抗炎作用,IL-10在人类动脉粥样硬化(Atherosclerosis, AS)的早期和进展中均有表达,并能抑制许多与动脉粥样硬化斑块形成、破裂和血栓形成有关的细胞进程。冠心病的病理基础是动脉粥样硬化和血栓形成。动脉粥样硬化的形成对损伤血管内膜而言既是免疫反应过程,又是炎性反应的过程。IL-10是主要的炎性调节因子,其分泌大约有75%由遗传因素决定,因此,IL-10基因可能与动脉粥样硬化及其并发症有关,是研究冠心病的一个有潜力的候选基因。本研究旨在探讨白细胞介素-10(IL-10)基因- 627位点多态性与早发冠心病和血清IL-10水平的相关关系。方法应用聚合酶链反应-限制性片断长度多态性(PCR-RFLP)分析方法,检测早发冠心病(CHD)患者163例和正常对照者112例IL-10基因-627位点多态性,采用ELISA法检测血清IL-10水平。结果IL-10基因-627位点基因型和等位基因频率在冠心病组和对照组之间无显著性差异,χ2值分别为1.9324, 1.5703, P>0.05。按性别分层分析IL-10基因型和等位基因频率,男性组χ2值分别为1.2708,0.8595,P>0.05;女性组χ2值分别为0.8254, 0.7127, P>0.05。血清IL-10水平在AA型、AC型和CC型之间存在显著性差异, P<0.05,但在冠心病组和对照组之间无显著性差异, P>0.05。结论IL-10基因-627位点多态性与中国人汉族人冠心病的易感性无显著关联,并且,这种关系不受性别因素的影响。但IL-10基因-627 A/C多态性可能影响IL-10基因的转录活性。更多还原

【Abstract】 Hypertension is a complex disorder that involves multiple genetic and environmental factors interacting to produce the characteristic phenotype. The pathogenesis of arterial thrombotic disease is very complex and involves multiple genetic and environmental factors related to atherosclerosis and thrombosis, as well as their interaction. Classically, acute thrombosis at the site of a ruptured, lipid-rich atherosclerotic plaque is understood as the precipitating event in the transition from stable or subclinical atherosclerotic disease to acute myocardial infarction (MI), ischemic stroke (IS), or peripheral arterial occlusion. To date, the prevention of arterial thrombotic disease has consisted of the modification of traditional cardiovascular risk factors that are largely environmental; however, approximately half of all thrombotic events occur in patients without such risk factors, and epidemiologic studies increasingly demonstrate that these are insufficient to explain completely the variations in incidence and risk.Some studies have shown that inherited risk factors contribute significantly to the development of coronary artery disease (CAD). Thus, in the current era of elucidation of the human genome, investigators have focused on the molecular genetics of thrombosis and atherosclerosis to improve their understanding of the pathobiology of arterial thrombosis.Increasing clinical and laboratory evidence suggests that hypertension per se may confer a prothrombotic or hypercoagulable state. Several genetic mutations affecting coagulation proteins have been suggested as prothrombotic risk factors. Among these genetic mutations, the thrombomodulin gene , transforming growth factor-beta-1 gene and interleukin 10 gene are an important potential candidate. This study aimed to estimate the proportion of incidence cardiovascular disease (CVD) among hypertensives that may be attributable to these gene polymorphisms.1. Thrombomodulin (TM) geneSoluble thrombomodulin (sTM) as abnormalities of levels of specific plasma markers of endothelial damage or dysfunction may be related to the complications of hypertension and the determination of blood pressure itself. We performed a case-control study, including 286 patients with essential hypertension(EH), 113 hypertensives with coronary arteries disease (CAD) and 213 age- and sex-matched controls. The TM -33G>A polymorphism was determined by polymerase chain reaction and restriction fragment length polymorphism (PCR–RFLP) analysis. TM on monocytes was measured by flow cytometry and sTM was determined with ELISA . Results We found that hypertensives have lower thrombomodulin on monocytes especially in patients with CVD. But we did not find significant difference in the sTM and the frequency of the A allele between hypertensives with CVD (14.2%) and controls (11%, P=0.616;OR 1.106, CI 0.746～1.640), or hypertensives (12.1%) and controls ( P=0.244, OR 1.330, CI 0.877～7.153). Similarly, the difference of the genotypic distributions could be neglected across the groups: GG :(GA/AA) was 81.2%:18.8% in controls, 79.7%:20.3% in hypertensives with CVD, and 77%:23% in hypertensives, respectively (vs. controls, all P>0.05). The lack of association also persisted after adjusting for other conventional risk factors. Conclusions Our results suggested that lower TM on monocytes associated with CVD risk in hypertensives, but no association of sTM with blood pressure. It also seemed not to support a significant association of the TM -33G>A polymorphism with CVD and EH in Chinese Han population. TM gene mutation may have a mildly affects on TM expression that might be balanced out by other factors during the progress of hypertention.2.Transforming growth factor-β1 (TGF-β1) geneEpidemiologic, clinical, and experimental evidence has suggested that upregulation of transforming growth factor-β1 (TGF-β1) may play a role in hypertensive disease. TGF-β1 is a multifunctional cytokine that regulates cell growth and differentiation, modulation of extracellular matrix and repair, which in turn regulate inflammatory and immune responses and can be involved in vascular remodelling. Transforming growth factor-β1 has been implicated in the pathogenesis of the vascular and target organ complications of hypertension. TGF-β1 may also regulate blood pressure via stimulation of endothelin-1 and/or renin secretion. Herein we explored the hypothesis that circulating levels of TGF-β1 protein are correlates of blood pressure levels. And study the relationship of the allele frequencies and genotype distribution of TGF-β1 gene polymorphism in patients with essential hypertension and coronary artery disease, and to analyze a association of the serum levels and genotype of TGF-β1 with essential hypertension and coronary artery disease. Methods The polymorphisms of TGF-β1 gene , including polymorphisms of TGF-β1 gene +869T/C and +915G/C, were analyzed by sequence specific primers-polymerase chain reaction ( PCR-SSP) methods in 286 patients with essential hypertension ,113 coronary artery disease and 213 healthy controls, and the serum level of TGF-β1 was determined by enzyme-linked immunosorbent assay( ELISA). Results The patients with coronary artery disease group showed significantly higher serum levels of TGF-β1 than essential hypertensives and control group. And essential hypertensives group showed significantly higher serum levels of TGF-β1 than control group (all P<0.05) the distributions of TGF-β1 gene +915G/C polymorphism did not show significant differences among patients with coronary artery disease, essential hypertensives and control group (P>0.05), but the TGF-β1 gene +869T/C polymorphism was significantly different between coronary artery disease group and control group(P<0.05). The relative risk of suffering from coronary artery disease in carriers of the C allele was 1.681 times than carriers of the T allele (OR =1.681, 95% CI: 1.213～2.328). the serum level of TGF-β1 C allele carriers was significantly higher than nocarriers. the serum level of TGF-β1 CC genotype was higher than TC genotype, and the serum level of TGF-β1 TC genotype was higher than TT genotype(CC:TC:TT was 7.06±0.69: 6.84±0.75: 6.12±0.58. It is suggested that the C allele may affect the TGF-β1 expression,even only one changed C allele of TGF-β1 gene +869 may influence the serum level of TGF-β1 expression. Conclusions TGF-β1 gene +869T/C polymorphism was associated with coronary artery disease, and C allele may be a risk factor for coronary artery disease in which the TGF-β1 C allele may be a increased risk factor by enhancing the TGF-β1 expression in the pathogenesis of essential hypertension with coronary artery disease. But it seemed not to support a significant association of the TGF-β1 gene +915 G/C polymorphism with coronary artery disease.3.Interleukin-10 geneInterleukin-10 (IL-10) is a cytokine produced by macrophages, T cells and B cells. It exerts an anti-inflammatory activity by inhibiting the production of pro-inflammatory cytokines and by activating B cells. IL-10 is expressed in both early and advanced human atherosclerotic plaques and inhibits many cellular processes involved in plaque progression, rupture, or thrombosis. The basic pathology mechanisms of coronary heart disease is atherosclerotic plaques and thrombosis. Interleukin 10 (IL10) is a major immunoregulatory cytokine . About 75% of the variability in IL10 secretion is determined by genetic differences. Therefore IL-10 gene seems to be implicated in both atherosclerosis and its acute complications. the IL-10 gene appears to be a good candidate for coronary heart disease studies. So our study aimed to investigate the relationship between interleukin-10 gene–627 polymorphisms and Serum interleukin-10 production and earlier coronary heart disease (CHD). Methods The genotype and allele frequency of interleukin-10 -627 gene site was assayed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). DNA samples were obtained from 163 patients with CHD and 112 controls. Serum IL-10 production was detected by ELISA method.Results No significant difference was found in the distribution of IL-10 genotype and allele frequency between healthy controls and patients with CHD.χ2 values were 1.9324 , 1.5703 respectively. P>0.05.Stratification analyses based on deferent sex, no significant difference was found in both male groups and female groups.χ2 values in male groups were 1.2708, 0.8595 and in female groups were 0.8254, 0.7127, respectively. P>0.05. Serum IL-10 production showed significant differences among AA genotype, AC genotype and CC genotype. But no significant difference was found between healthy controls and patients with CHD. Conclusions Our results suggest that IL-10–627 A/C polymorphisms are not associated with an increased risk of CHD, but it might have a role on IL-10 gene expression.更多还原