【作者】 邓烈华；

【导师】 许顶立；

【作者基本信息】 南方医科大学 ， 心血管病学， 2009， 博士

【摘要】 【研究背景】收缩期高血压（ISH）是以收缩压增高和脉压差增大为特点的一种特殊类型高血压,常见于老年人,具有较高的致死、致残率,已成为人们研究的热点。随着我国人口老年化进程的加剧和人群疾病谱的变化,ISH患病率逐年增加,ISH作为原发性高血压的一种特殊亚型,发病机制复杂且远未阐明,目前认为是遗传因素和环境因素共同作用的结果。随着分子遗传学的发展,遗传因素在原发性高血压发病机制中的地位越来越受到重视,迄今为止被研究过的高血压候选基因至少有150种,它们涉及肾素-血管紧张素-醛固酮系统、交感神经系统、下丘脑-垂体轴、内皮素、利钠肽、激肽释放酶-激肽系统、类固醇激素、前列腺素、生长因子、激素、骨架蛋白、黏附分子、细胞内信使、脂质代谢、糖代谢、载脂蛋白、离子通道和转运体等,但各家报道结果不一致,还没有一个基因或基因变异被肯定与原发性高血压相关。原发性高血压等复杂性状疾病有以下共同特点:遗传模式未确定、遗传异质性强、外显率低、多个基因参与、单一基因的作用微弱等。虽然血压升高是高血压患者的共同表型,但患者的发病机制和与之相关的基因变异千差万别。因此对遗传异质性很强的高血压群体进行基因多态性分析,尤其当样本量不够大时,研究结果不一致就不足为奇了。我们认为在目前诊断的原发性高血压患者中可能包括了太多的亚型,笼而统之的研究难以获得结果。选择一些特殊的亚型进行研究可能成为阐明高血压发病机制的突破口。动脉僵硬度增加、顺应性降低被认为是ISH典型的病理生理学特点,通过构建ISH模型人们发现促进动脉硬化的因素都可能导致ISH的发生发展。近来研究表明,弹性蛋白在大动脉中层中的含量和结构的改变都能导致动脉弹性降低,僵硬度增加。特别是弹性蛋白基因改变在血管发育和血管疾病中作用的研究引起了我们的极大兴趣,弹性蛋白基因的突变（mutation）是导致主动脉瓣上狭窄的关键,而Williams综合征则是由于弹性蛋白基因缺失（deletion）所致。Li等应用转基因小鼠的研究发现,染色体7q11.23的弹性蛋白基因完全缺失小鼠(Eln^-/-)因内皮下细胞增殖和平滑肌细胞改变引起阻塞性动脉疾病而死亡,存活时间不长;而弹性蛋白基因单倍体缺失小鼠(Eln^+/-)的特征与人SVAS患者的动脉血管改变极其相似,表现为动脉弹性蛋白膜（层）变薄和平滑肌细胞层数目增加。Faury等进一步研究还发现Eln^+/-小鼠出生后呈持续高血压状态,平均动脉压较野生型小鼠高20-30mmHg,提示弹性蛋白基因可能是原发性高血压的发病基因之一。国外众多研究还表明,弹性蛋白基因多态性及单体型与动脉僵硬度密切相关,Hanon等研究表明弹性蛋白SNP位点（rs2071307,exon16）与老年患者的弹性动脉的舒张性密切相关。Hiroyuki等认为弹性蛋白SNP位点（rs34208922,3’-UTR（+502）A）的基因变异可使弹性蛋白mRNA的表达下调,从而导致动脉壁弹性蛋白含量降低及功能的改变。Iwai等进一步证实该位点的变异与动脉僵硬度及收缩压升高密切相关。还有研究表明弹性蛋白基因多态性（rs41350445 and SNP2 rs41500150 in exon 5,rs34945509 in exon 20）可能在家族性颅内动脉瘤和蛛网膜下腔出血的发生、发展过程中起重要作用,其作用机制为以上SNP位点基因变异导致颅内动脉僵硬度增加,动脉顺应性降低所致。因此有理由推测弹性蛋白的基因多态性在汉族人群收缩期高血压的发生、发展过程中也可能发挥重要作用。脉搏波是心脏的搏动沿动脉血管和血流向外周传播而形成的,其传播速度主要决定于动脉管的物理特性和几何特性,尤其是动脉管壁的弹性特性。动脉血管弹性越大,脉搏波传播速度越小。临床上常以臂-踝脉搏波速度（baPWV）作为评估动脉血管弹性的定量指标。研究表明,在原发性高血压患者人群中,在舒张压不变的情况下,baPWV随收缩压升高而显著增快,在收缩压不变的情况下,baPWV随舒张压升高,而显著降低。C反应蛋白作为一种炎性标志物,研究表明,其与动脉僵硬度以及PWV密切相关,在动脉硬化的发生发展过程中发挥重要作用,其作用机制可能与C反应蛋白能使大动脉中层中弹性蛋白的合成与降解失衡,导致弹性蛋白的结构和功能的改变,从而促使动脉硬化。因此,与ISH密切相关的弹性蛋白基因单核苷酸多态性也将与PWV及CRP密切相关。本课题拟研究弹性蛋白基因5个SNP位点（SNP1 rs41350445 and SNP2rs41500150 in exon 5,SNP3 rs2071307 in exon16,SNP4 rs34945509 in exon 20,SNP5 rs34208922 in 3’-UTR）与收缩期高血压的关系,并探讨其对收缩期高血压患者脉搏波传导速度（PWV,动脉僵硬度的评价指标）、颈动脉中层厚度（IMT）以及C反应蛋白（CRP）的影响,从而确定与收缩期高血压密切相关又具有临床意义的SNP位点,揭示收缩期高血压的遗传背景。第一章收缩期高血压患者脉搏波传导速度与心率变异性的研究目的研究收缩期高血压患者脉搏波传导速度与心率变异性的特点以及两者在评估收缩期高血压患者心血管风险方面的作用。方法选取89例收缩期高血压患者及98例双期高血压患者,应用SUN-8800动脉硬化检测仪检测颈-股动脉脉搏波传导速度（cfPWV）及颈-桡动脉脉搏波传导速度（crPWV）,采用5分钟短程频域分析法分析其心率变异性（HRV）指数[总功率（TP）、极低频功率（VLF）、低频功率（LF）、高频功率（HF）以及低频功率与高频功率之比（LF/HF）],观察收缩期高血压患者与双期高血压患者间cfPWV、crPWV及HRV指数的差异,采用偏相关分析cfPWV、crPWV与HRV指数的相关性。再对89例收缩期高血压患者进行总体心血管风险评估,根据危险程度分为低危（n=3）、中危（n=17）、高危（n=35）以及极高危（n=34）四组,观察收缩期高血压患者不同危险程度组间cfPWV、crPWV及HRV指数的差异以及它们与心血管危险程度的相关性。结果收缩期高血压组的cfPWV明显高于双期高血压组[收缩期高血压:（399.6±48.2）cm/s比双期高血压:（374.3±39.7）cm/s,P＜0.05],而两组crPWV差异无统计学意义[收缩期高血压:（371.5±37.5）cm/s比双期高血压:（363.5±31.5）cm/s,P＞0.05];收缩期高血压组的心率变异指数VLF、LF以及LF/HF高于双期高血压组[收缩期高血压:（4.35±1.07）log ms²、（3.28±0.86）logms²、（1.19±0.53）比双期高血压:（3.78±0.82）log ms²、（2.93±0.59）logms²、（0.89±0.43）,P＜0.05]而TP、HF低于双期高血压组[收缩期高血压:（4.76±1.03）log ms²、（2.75±1.43）log ms²比双期高血压:（5.15±1.36）log ms²、（3.27±1.57）log ms²,P＜0.05]。cfPWV与VLF、LF呈显著正相关（偏相关系数分别为0.563,0.549,P均＜0.05）而与TP、HF呈显著负相关（偏相关系数分别为-0.542,-0.571,P均＜0.05）,crPWV与VLF、LF、HF及TP无明显相关性。收缩期高血压患者总体心血管风险程度与cfPWV及crPWV均呈显著正相关（Spearman相关系数分别为0.765,0.563,P均＜0.04）,cfPWV随风险程度增高而显著增加（P＜0.05）;总体心血管风险程度与TP、HF呈显著负相关（Spearman相关系数分别为-0.467,-0.421,P均＜0.03）,而与VLF、LF、LF/HF呈显著正相关（Spearman相关系数分别为0.438,0.479,0.126,P均＜0.04）,VLF、LF、LF/HF随风险程度增高而显著增加（P均＜0.01）,但TP、HF随风险程度增加而显著降低（P均＜0.01）。结论收缩期高血压患者的颈-股动脉脉搏波传导速度及心率变异指数VLF、LF、LF/HF显著增高而心率变异指数TP、HF显著降低,颈-股动脉脉搏波传导速度与心率变异性指数可作为评价收缩期高血压患者心血管危险程度的重要指标。第二章收缩期高血压患者脉搏波传导速度与微量白蛋白尿的相关性研究目的研究收缩期高血压患者脉搏波传导速度（PWV）的变化对微量白蛋白尿（MAU）的影响,探讨PWV与MAU的相关性。方法应用SUN-8800动脉硬化检测仪检测265例收缩期高血压患者颈-股动脉脉搏波传导速度（cfPWV）及颈-桡动脉脉搏波传导速度（crPWV）,采用免疫比浊法测定患者24小时尿微量白蛋白含量,根据微量白蛋白尿检测结果分为MAU阴性组（n=116）和MAU阳性组（n=149）,比较MAU阳性组与MAU阴性组cfPWV、crPWV以及各种心血管事件发生率之间的差异,采用趋势检验分析PWV的变化对MAU的影响,并用直线相关和logistic回归分析PWV与MAU间的相关性。结果在控制年龄、收缩压及脉压的后MAU阳性组的cfPWV及crPWV均高于MAU阴性组（cfPWV:393.5±28.7cm/s vs 379.4±25.2cm/s,P＜0.001;crPWV:387.6±27.1 vs 368.5±23.6,P＜0.001）;MAU阳性组总体靶器官损害、左室肥厚、心肌梗死、外周血管疾病以及肾功能不全发生率高于MAU阴性组（P＜0.05）;在控制年龄、收缩压、舒张压、脉压以及心率的情况下微量白蛋白尿阳性的危险性随PWV的增加而显著增加;cfPWV、crPWV与24小时尿白蛋白排泄量均呈显著正相关,相关系数分别为:r=0.875,P=0.001;r=0.763,P=0.017。logistic回归分析显示,在控制年龄、收缩压以及脉压的基础上cfPWV与crPWV均与24小时尿白蛋白排泄量呈独立相关（P=0.015,P=0.023）。结论PWV增加可增加MAU的危险性,cfPWV、crPWV是MAU独立危险因素。第三章弹性蛋白基因单核苷酸多态性与收缩期高血压关系的研究目的研究弹性蛋白基因单核苷酸多态性与收缩期高血压的关系,探讨弹性蛋白基因单核苷酸多态性对收缩期高血压患者脉搏波传导速度、颈动脉中层厚度以及C反应蛋白的影响。方法选取358例收缩期高血压患者（ISH组）,413例原发性高血压患者（EH组,非收缩期高血压）以及年龄相匹配的244例健康人群（NT组）,采用链接酶检测反应（LDR-PCR）测定各患者5个SNP位点（SNP1 rs41350445 and SNP2 rs41500150in exon 5,SNP3 rs2071307 in exon16,SNP4 rs34945509 in exon 20,SNP5rs34208922 in 3’-UTR）的基因表型,采用动脉硬化装置检测桡-踝脉搏波传导速度（baPWV）,采用超声技术测定颈动脉中层厚度（IMT）,采用ELISA法检测患者外周血清中的C反应蛋白（CRP）浓度。结果弹性蛋白基因单核苷酸多态性（SNP rs34208922）与收缩期高血压显著相关,ISH组A等位基因频率高于EH组和NT组(0.068 vs 0.036 vs0.014;P＜0.001,P_corr＜0.005)。弹性蛋白基因单核苷酸多态性（SNP rs2071307）也可能与老年单传收缩期高血压相关,ISH组A等位基因频率高于NT组(0.103 vs 0.047,P=0.002,P_corr=0.01),但ISH组A等位基因频率与EH组等位基因频率差异无统计学意义(0.103 vs 0.079,P=0.068,P_corr=0.062)。弹性蛋白基因单核苷酸多态性（SNP rs34208922,SNP rs2071307）与baPWV和CRP显著相关,baPWV和CRP随以上SNP位点的A等位基因频率的增加而增高（P for trend=0.015,P for trend＜0.001,P for trend=0.001,P for trend=0.017）,其他3个基因单核苷酸多态性与ISH、baPWV、CRP和IMT相关性无统计学意义。单体型TGGTA和TGAT-与收缩期高血压显著相关(P=0.0001,P_corr=0.0021;P=0.0023,P_corr=0.0483)。结论弹性蛋白基因变异可显著增加我国汉族人群发生收缩期高血压以及动脉硬化的危险性。更多还原

【Abstract】 BackgroudIsolated systolic hypertension of the eldly was a subtype of Essential hypertension and characteristiced by elevated systolic and pulse pressure,high mortality and high morbility.Even through,ISH has been an outcome of co-contribution of environment and inherit factors,unfortunately,the pathogenesis of ISH has not been fully understood.With the development of molecular genetics, the effect of inherit factor on the Essential Hypertension morbility has been emphasized.As yet,150 candidate genes for EH had been studied,which involved in renin-angiotensin-aldosterone system（RAAS）,sympathetic nervous system（SNS）, low-cerebral ganglion-master gland-axon,endothelin（ET）,natriuretic peptide, KAL-kinin system,steroid hormone（STH）,prostaglandin（PG）,growth factor（GF）, hormone,skeleton protein,adhesion molecule,intracellular messenger,lipide metabolism,glycometabolism,apolipoprotein（APO）,ion channel,and so on.But no gene variant has been confirmed to be associated with EH.Owing to characteristic of the indefinite heredity model,high genetic heterogeneity,low penetrance,more genes participated,single gene effect feeble for the EH as some polygene diseases,and relatively low samples for the studies,it was all in the day’s work that the studies arrived at different results.Because of the EH including some subtypes,it is difficult to get confirmedly information about the gene association with EH,and may be as a breakthrough to interpret EH pathogenesis to chose a certain subtype.ISH was characterized by high artery stiffness and low artery compliance,it is reported that all the factors involved in promoting artery stiffness which might be lead to ISH.It had been confirmed that the change of structure and function of elastin located in the large artery media could caused artery stiffness,and some reports about the contributions of elastin to angio-development and cardiovascular diseases revoked our interest in studying elastin gene.a deletion involving 7q11.23 that results in hemizygosity of the elastin gene has been identified as the mechanism responsible for the Williams and Buren syndrome,which is characterized by supravalvular aortic stenosis,hypertension or peripheral stenoses.Li et al had reported that elastin is an essential determinant of arterial morphogenesis,the characterization of mice haploinsufficient for elastin(Eln^+/-) revealed a role for elastin in the formation of vessel wall structure.The arteries of Eln^+/- mice exhibited thinner elastic lamellae and an increased number of smooth muscle cell layers.Most interestingly,these identical changes have been observed in the arteries of patients with SVAS.D’Armiento J demonstrated that mice haploinsufficient for elastin develop structural changes in vessel walls similar to those seen in patients with mutations in the elastin gene.due to mechanical changes in the vessel wall,these animals exhibit increased mean arterial pressures.The results evoke the possibility that alterations in elastin may contribute to the development of essential hypertension in patients.Some studies had also reported that elastin gene polymorphisms were associated with artery stiffness,Hanon O et al.identified the relationship between the Ser422Gly polymorphism of the elastin gene in exon 16（rs2071307） and the distensibility of elastic arteries.9 Interestingly,Iwai N et al.revealed the association of the rs34208922 polymorphism with hypertension and pulse wave velocity.10 Furthermore,Akagawa H et al.reported that SNP（rs34208922） is involved in the reduction of elastin mRNA transcripts in vitro and ex vivo.11 Recently,elastin has also been suggested to be a potential candidate gene for familial intracranial aneurysms（IAs） 12,13and subarachnoid hemorrhages（SAH）,14 which we also found defects in the internal elastic lamina.As a result,ELN may be considered as a functional candidate gene for ISH.Pulse wave velocity（PWV） is a known marker of arterial stiffness and defined as the speed in which the pulse wave travels along the length of an artery.It was reported that at any given SBP,brachial-ankle Pulse wave velocity（baPWV） decreased with the increasing DBP,and at any given DBP,baPWV increased with the increasing SBP in Chinese patients with essential hypertension.Also,baPWV was higher in patients with ISH compared to age-matched healthy individuals.Serum C-reactive protein（CRP） is known as a marker of inflammation and some studies have shown that inflammation is independently associated with arterial stiffness and PWV.Alternatively,inflammation may induce structural changes in the arterial wall by altering the balance between elastin breakdown and synthesis.Therefore,the functional candidate gene for ISH may be associated with baPWV and CRP.The main purpose of this study was to evaluate the possible association between polymorphism of the ELN gene and ISH,baPWV,CRP and IMT.Part oneThe study of Pulse Wave Velocity and Heart Rate Variability in patients with Isolated Systolic HypertensionObjective To investigate the characteristics of Pulse Wave Velocity（PWV） and Heart Rate Variability（HRV） and the effect of them on estimating the Cardiovascular Risk in patients with isolated systolic hypertension（ISH）.Methods 89 ISH patients and 98 systolic-diastolic hypertension patients admitted in our hospital were taken carotid-radial PWV（crPWV）,carotid-femoral PWV（cfPWV） and HRV（total power（TP）,very low frequency（VLF）,low frequency（LF）,high frequency（HF） and LF/HF,analyzed by 5min short-term frequency domain） by SUN-8800,and then survey the difference of them between the ISH patients and systolic-diastolic hypertension patients and the correlation of PWV with HRV. Furtherly,89 ISH patients were divided into four groups:low risk group（n=3）, moderate risk group（n=17）,high risk group（n=35） and very high risk group（n=34） according to the degree of Total Cardiovascular Risk and investigate the difference of cfPWV,crPWV and HRV among the four groups and the correlation of cfPWV, crPWV and HRV with the degree of Total Cardiovascular Risk. Results The cfPWV of ISH group is significantly higher than that of sys-diastolic hypertension group(ISH:399.6±48.2cm/s vs sys-diastolic hypertension: 374.3±39.7cm/s,P＜0.05],but there is no difference in crPWV between ISH group and sys-diastolic hypertension group（ISH:371.5±37.5cm/s VS sys-diastolic hypertension:363.5±31.5cm/s,P＞0.05）.The VLF,LF and LF/HF in ISH group are markedly higher than those in sys-diastolic hypertension group（ISH:4.35±1.07logms², 3.28±0.86logms² and 1.19±0.53 vs sys-diastolic hypertension:3.78±0.82logms², 2.93±0.59logms³ and 0.89±0.43,P＜0.05）,while the TP and HF are evidently lower than those in sys-diastolic hypertension group（ISH:4.76±1.03logms², 2.75±1.43logms² vs sys-diastolic hypertension:5.15±1.36logms,3.27±1.57logms², P＜0.05）.The cfPWV is positively related with the VLFand LF and negatively related with the TP and HF（r=0.563,r=0.549,r=-0.542 and r=-0.571,respectively,P＜0.05） but the crPWV is not related with VLF,LF,TP and HF.The cfPWV and cfPWV are markedly positive correlated with the degree of the Total Cardiovascular Risk（r_s=0.765,r_s=0.563,P＜0.05）,with the increase of degree of the Cardiovascular Risk,the cfPWV is significantly increased（P＜0.05）,but the crPWV is not significantly increased（P＞0.05）.The VLF,LF and LF/HF are markedly positive correlated with the degree of the Total Cardiovascular Risk（r_s=0.438,r_s=0.479 and r_s=0.126,P＜0.05）,adversely,the TP and HF are dramaticly negative correlated with the degree of the Total Cardiovascular Risk（r_s=-0.467,r_s=-0.421,P＜0.05）, with the increase of degree of the Cardiovascular Risk,the VLF,LF and LF/HF are significantly increased,but the TP and HF are markedly decreased（P＜0.01）.Conclusion The cfPWV,VLF,LF and LF/HF are significantly increased,however, the TP and HF markedly decreased in ISH patients.cfPWV and HRV should be as important clinic indexs to evaluate ISH cardiovascular risk and the effect of ISH treatment. Part twoThe Association of Pulse Wave Velocity and Microalbuminuria in Older Patients with Isolated Systolic HypertensionObjective To investigate the effects of change of pulse wave velocity（PWV） on microalbuminuria（MAU） and analyze the association between PWV and MAU in patients with Isolated systolic hypertension.Methods Carotid-radial PWV（crPWV） and carotid-femoral PWV（cfPWV） were determined in 265 patients with Isolated systolic hypertension.The participants were assigned to groups with microalbuminuria and with normoalbuminuria,and their characteristics,cfPWV,crPWV and the morbidity of cardiovascular incidence were compared.We also examined the association between MAU and cfPWV,crPWV using linear correlation and logistic regression analyses.Results 149 microalbuminuria participants had significantly higher cfPWV,crPWV and morbidity of cardiovascular incidence than those with normoalbuminuria.After adjusted the age,systolic blood pressure,diastolic blood pressure and pulse pressure, the Odds Ratio of MAU were increased with the increasing of cfPWV and crPWV among the quartiles of cfPWV and crPWV（P for trend=0.017,P for trend=0.025）. Linear correlation analyses showed that cfPWV and crPWV were markedly positive correlated with the 24 hours urinary albumin excretion（r=0.875,P=0.001;r=0.763, P=0.017）,multiple logistic regression analyses showed that cfPWV and crPWV were independently associated with MAU（P=0.015,P=0.023） after adjustment for age, systolic blood pressure,diastolic blood pressure and pulse pressure.Conclusion MAU was independently associated with cfPWV and crPWV,cfPWV and crPWV were the important affected factors of MAU. Part threeA Study on Polymorphisms of Elastin Gene in Chinese Han Patients with Isolated Systolic HypertensionBackground.Elastin（ELN） is mainly located in the internal elastic lamina of large arteries.Degradation of elastin is expected to induce large vessel stiffness,which could lead to elderly systolic hypertension.Recent studies have shown the polymorphism of ELN is associated with stiffness of elastic arteries and elevated blood pressure,however,there are no further studies on isolated systolic hypertension.Methods.We identified the genotype of the ELN gene in 358 patients with isolated systolic hypertension（ISH）,413 essential hypertension patients with elevated diastolic blood pressure（EH） and 244 age-matched normotensive（NT） controls for 5 SNPs and detected the baPWV,CRP and IMT for these patients.Results.ISH was statistically significant in association with SNP rs34208922(A allele frequency was 0.068 in ISH patients,0.036 in EH patients and 0.014 in NT controls;P＜0.001,P_corr＜0.005) and possibly with SNP rs2071307(A allele frequency was 0.103 in ISH patients,0.079 in EH patients and 0.047 in NT controls; P=0.002,P_corr=0.01),however,the A allele frequency was not different between ISH patients and EH patients.In addition,baPWV and CRP were significantly associated with SNP rs34208922 and rs2071307.The other three SNPs were not significantly associated with ISH,baPWV,CRP or IMT.Haplotypes of TGGTA and TGAT-were also significantly associated with ISH(P=0.0001,P_corr=0.0021;P=0.0023, P_corr=0.0483).Conclusions.Variants within the elastin gene are associated with increased risk of ISH and aortic stiffness in the Chinese Han population.更多还原