冠心病与心肌重塑的新的危险因素：同型半胱氨酸硫内酯复合物及其代谢酶基因多态性与生长分化因子15的基因多态性

【作者】 杨旭；

【导师】 惠汝太；

【作者基本信息】 中国协和医科大学 ， 遗传学， 2008， 博士

【摘要】 第一部分中国人群同型半胱氨酸硫内酯复合物及其代谢酶同型半胱氨酸硫内酯酶基因多态性对冠心病的影响背景血浆同型半胱氨酸(Homocysteine,Hcy)水平升高已被证明是冠心病的独立危险因素,但致病机制尚未完全明了。目前认为,同型半胱氨酸硫内酯(Homocysteine thiolactone,HTL)作为同型半胱氨酸的代谢产物之一,可以通过修饰蛋白表面的赖氨酸残基,破坏蛋白质的结构并损伤其生理活性,形成HTL复合物。研究表明,在Hcy致冠心病过程中HTL复合物发挥了重要作用,可能是Hcy致冠心病的关键因素之一,因此近年来HTL复合物作为心血管疾病的一个标志物而日益受到关注。目前国内临床上普遍开展的检测方法为检测总同型半胱氨酸(tHcy)含量,尚未有准确测定同型半胱氨酸硫内酯复合物的方法。国外近年来开展了用HPLC方法测定HTL复合物的方法,但价格较高,难以普及。本研究首先建立稳定可靠、简便易行的测定HTL复合物的方法,通过检测血浆HTL复合物水平系统地探究HTL复合物与冠心病的关系,以期明确HTL复合物是否为中国人群冠心病发病的危险因素。然后应用多聚酶链限制性片断多态反应技术对paraoxonase-2进行基因分型,并探讨paraoxonase-2基因多态性与冠心病的关系,为进一步深入广泛研究提供有益线索。材料与方法1.通过用HTL体外修饰白蛋白,得到HTL复合物抗原,并用HTL复合物抗原免疫新西兰大白兔,得到抗HTL复合物抗体,依据Eric Ferguson及Yosltinori Uji等的方法学描述,建立竞争性ELISA方法以检测血浆HTL-HSA复合物。2.样本组:选择2002年1月至2003年12月我院心内科住院的患者共254例,经冠状动脉造影证实患冠心病;对照组:选择正常查体人群308例为对照组。上述所有入选对象均无肝、肾、肿瘤、血液、风湿病等疾病,已知其它原因的心脏病亦被排除,调查内容包括冠心病史、高脂血症史、吸烟史及饮酒史等。计算体重指数(BMI),采用ELISA方法检测HTL复合物,采用HPLC的方法进行Hcy的测定,采用全自动生化分析仪进行血糖、生化、血脂等血生化指标的测定。3.通过低渗溶血法分离白细胞,盐氯法提取人基因组DNA,应用多聚酶链反应—限制性内切酶片断长度多态性技术(PCR-RFLP)分析Paraoxonase-2基因311位Cys-Ser多态性。4.采用SPSS 10.0软件进行统计学处理,计量资料以Mean±SD表示,各组间数据的比较依据资料的性质,采用t检验、x~2检验。采用多元逐步logistic回归分析、简单直线相关分析及多元线性回归分析。等位基因确认符合Hardy-Weinberg平衡。研究结果1.得到HTL修饰的人血清白蛋白及兔血清白蛋白。并通过DTNB法及TNBS方法鉴定修饰结果。TNBS方法显示BSA及RSA表面约20%的赖氨酸残基被HTL修饰。2.得到抗HTL复合物多抗且抗体的工作浓度约为1:500。采用了竞争性固相ELISA法鉴定所得抗体是特异性抗HTL复合物的抗体,且HTL复合物的组分包括HTL修饰血中存在的多种蛋白形成的相应HTL复合物,如HSA、HB、LDL及HDL等。3.建立ELISA方法测定HTL复合物含量。本方法特异性良好,特异性抗HTL修饰后蛋白即HTL复合物;灵敏度良好,检测范围在12.5—200u/ml之间,若以正常人群90%以上的值37.57 u/ml确定为正常上限值,此点位于曲线中部,灵敏度最高。精密度测定结果显示:批内变异系数为6.4%,批间变异系数为8.9%,实验重复性良好。4.冠心病组及其各组分与对照组HTL复合物水平的比较:冠心病组HTL复合物水平明显高于对照组(40.65±10.87 u/ml vs.30.58±10.20 u/ml,P＜0.01),多元logistic回归分析显示,HTL复合物对冠心病的相对危险度(OR)为7.34,(95%可信区间4.020～13.406,P＜0.01)。HTL复合物与冠状动脉血管堵塞程度具有直线关系:1、2和3支病变分别为:35.59±10.34 units/ml(n=76);41.88±8.83(n=70)and 43.13±11.47(n=108)(r=0.174,P＜0.01)。5.冠心病组paraoxonase-2基因311位点C等位基因频率显著高于对照组(0.232 vs.0.149,P=0.017),CC基因型的频率为7.48%,显著高于对照组1.62%(OR)为4.367(95%可信区间:1.178 to 16.191,P＜0.01)。结论HTL复合物可能是预测心血管病事件的生化指标,检测血浆HTL复合物水平对冠心病的预防、诊断及治疗具有重要临床价值。paraoxonase-2基因311位CC基因型是冠心病患病的风险标志物。第二部分生长分化因子15的基因多态保护原发性高血压患者左心室重塑背景生长分化因子15(Growth-differentiation factor 15,GDF15)是心脏中一个新的抗肥厚因子。当心肌受损,GDF15被诱导表达,并在左心室肥厚性生长的病程中发挥重要的调控作用。我们假设GDF15的基因多态可能与原发性高血压患者的左室重塑相关。方法我们用随机整群抽样法从中国农村的社区人群中入选了1527名(506名男性,1021名女性)原发性高血压患者,用M型超声心动图检测左心室重量和结构,用聚合酶链式反应—限制性内切酶长度多态性法进行基因多态分型,分析了GDF15的2个标签SNP(tagSNP)(-3148C/G,+157A/T)和1个功能性SNP位点(+2438C/G)与高血压继发左心室重塑的关联。同时利用荧光素酶报告系统和ELISA方法检测血浆中GDF15水平,对GDF15的promoter区功能SNP进行了研究。结果在GDF15的三个多态位点中,只有位于启动子区的标签SNP-3148C/G与舒张压、左心室肥厚和超声指标相关联。具体说来,-3148G等位基因分别与较低的舒张压(CC,CG和GG,舒张压分别为100.0±12.6 mmHg,98.3±11.7 mmHg.97.6±11.5mmHg,P=0.015)和较低的左室肥厚程度相关联(OR=0.78,95%CI 0.65-0.94,P=0.009)。在多元回归分析中,-3148G是左室舒张末期直径(β=-0.10,P=0.0001)、收缩末期直径(β=-0.09,P=0.0007)、左室重量(β=-0.11,P＜0.0001)和左室重量指数(β=-0.13,P＜0.0001)的独立预测因子。-3148G对左室结构和重量的预测独立其他传统危险因素,包括性别、年龄、体表面积、血压、糖尿病、抗高血压治疗、吸烟和饮酒等。线性回归分析表明-3148G的作用具有显著的基因量效关系。在荧光素酶报告系统中-3148G比-3148C荧光素酶活性显著升高192%(P＜0.001),在心肌肥厚患者中-3148G与血浆水平的GDF15的升高也具有明显相关性(p=0.04)。结论GDF15基因启动子区的遗传多态性与原发性高血压患者的左心室大小、重量和肥厚程度紧密相关。更多还原

【Abstract】 BackgroundElevated levels of serum homocysteine(Hcy) are an independent risk factor for coronary heart disease(CHD).However,the precise mechanism of homocysteine-associated cardiovascular disease is still unclear.Available data suggest that homocysteine thiolactone(HTL),as one of metabolites of Hcy,can react with lysine residues in proteins forming HTL adducts,damaging their structure and impairing their physiological activities.The most important pathogenesis of HTL adducts includes:1. harmful to human endothelial cell and adversely affecting vascular endothelium; 2.stimulating aggregation of low density lipoprotein(LDL),inducing LDL uptake by macrophages in culture and facilitating the formation of foam cells;3.HTL modified LDL has been found to be easier to form small dense LDL,one of the most important risk factors for CHD;4.HTLresult in the formation of thromboxane B2 and prostacyclin 6-keto-PGF1 alpha during thrombosis,which induces primary platelet aggregation and clot formation.HTL adducts are now considered as markers for cardiovascular diseases. Homocysteine thiolactone hydrolase(HTase),which is a calcium-dependent enzyme, may hydrolyze HTL to Hcy and thereby prevent the homocysteinylation of proteins. Experiments in vitro demonstrate that high activity forms of HTase afford better protection against protein homocysteinylation than low activity forms of HTase.Our research aimed to develop a stable and convenient assay system for measuring HTL adducts,and to evaluate the correlation between plasma HTL adducts and CHD. Homocysteine thiolactone adducts have been proposed as the culprit of homocysteine related cardiovascular diseases.We studied the association of these adducts in plasma, and the gene polymorphism ofparaoxonase-2 with CHD.Materials and Methods1.The antigen of HTL adducts was prepared by modifying the rabbit albumin in vitro.Then female New Zealand White rabbits were immunized using standard protocols. Special antibody against HTL adducts were developed and evaluated.An ELISA method was established for measuring HTL adducts;2.Two hundred and fifty-four patients and 308 controls were recruited for the study. HTL adducts were determined with ELISA.Detailed medical history and thorough physical examinations were performed on all participants.Weight,height and the body mass index(BMI) were recorded.The level of plasma HTL adducts was measured by ELISA method and plasma Hcy was detected by HPLC methods.Blood biochemical analyses were carried out by using automatic biochemistry analyzer.3.The codon 311 polymorphism of paraoxonase-2 gene was genotyped by using polymerase chain reaction and restrictive digestion.4.Statistical analysis:All calculations used SPSS 10.0 for Windows statistical analysis software.Measurement data was indicated by Mean±SD.Data among groups was comp rated by t-test or x~2 test according to the type of variables.Multiple stepwise logistic regression analysis、correlation analysis and multiple linear regression analysis were used.The distribution of the HTase gene polymorphism was in Hardy-Weinberg equilibrium.Results1.Human serum albumin and rabbit serum albumin were modified by HTL.The extent of modification was determined by DTNB and TNBS assays.TNBS assay showed that 20 percent of lysine residues of HSA and RSA were modified by HTL.2.Polyclonal antibody directed against HTL adducts was generated and its working concentration is about 1:500.In order to determine antibody specificity solid phase competition-based ELISA techniques were used.We conclude that the antibody we got is specifically directed against protein modified by HTL and not against native protein.3.The specificity and the sensitivity of the methods allow detection of HTL adducts quantities from 12.5 to 200 u/ml.High plasma HTL adducts was defined based the 90th percentile of the value of the control population(≥37.57 u/ml).Intra-assay coefficient of variation was 6.4%and inter-assay coefficient of variation was 8.9%.4.The plasma level of HTL adducts were significantly higher in patients than in controls(40.65±10.87 u/ml vs.30.58±10.20 u/ml,P＜0.01),with odds ratio 7.34,(95% confidence interval 4.020～13.406,P＜0.01),and increased according to the number of atherosclerotic coronary arteries:35.59±10.34 units/ml(n=76);41.88±8.83(n=70) and 43.13±11.47(n=108) in subjects with 1,2 and 3 affected arteries,respectively(r=0.174, P＜0.01).5.The frequency of CC genotype was significantly higher in patients with coronary heart disease(7.48%) than in controls(1.62%,P＜0.01),with adjusted odds ratio of 4.367 (95%confidence interval:1.178 to 16.191,P＜0.01),so was the C allele(23.2%vs. 14.9%,P=0.017).ConclusionHigh plasma HTL adducts and the CC 311 genotype of paraoxonase-2 gene may be the emerging risk factor for CHD. Background Growth-differentiation factor 15(GDF15) is a novel antihypertrophic factor in the heart,which is induced in response to cardiac injury and plays important regulatory role in the process of left ventricular hypertrophy.We hypothesized that genetic variants of GDF15 may associate with left ventricular mass and geometry in hypertension.Methods A community-based hypertensive population sample of 1527 persons was studied by Mono-mode echocardiography.Three single-nucleotide polymorphisms (SNPs),including one tagSNP -3148C＞G of a natural haplotype and two exonic SNP (+157A＞T and +2438C＞G) were genotyped.The SNP functions were studied by use of luciferase reporter assays and determination of GDF-15 serum levels.Results Only the tagSNP -3148G showed significantly association with lower risk of left ventricular hypertrophy(OR=0.78,95%CI 0.65-0.94,P=0.009).In multiple regression analyses,-3148G predicted statistically significant decrease in left ventricular end-diastolic diameter(β=-0.10,P=0.0001),end-systolic diameter(β=-0.09,P=0.0007), mass(β=-0.11,P＜0.0001) and indexed mass(β=-0.12,P＜0.0001).These effects were independent of conventional factors,including sex,age,body surface area,blood pressure,diabetes,and anti-hypertensive treatment,and cigarette and alcohol consumption.The SNP -3148G-associated GDF-15 promoter exhibited 192%(P＜0.001) of transcription activity than the SNP -3148C-associated promoter.The -3148G allele was also associated with a significant increase of GDF-15 serum level(P=0.04) in the hypertensive subjects.Conclusions—Genetic variation within the promoter region of GDF15 gene is strongly associated with left ventricular size,mass and hypertrophy status in human essential hypertension.更多还原