【作者】 曾国良；

【导师】 彭健；

【作者基本信息】 南方医科大学 ， 心血管内科， 2009， 硕士

【摘要】 一、研究背景P-选择素(又称为颗粒膜蛋白GMP140,血小板激活依赖性颗粒外膜蛋白PADGEM或CD62)是一种黏附分子,属于选择素(selectin)家族,主要表达于激活的内皮细胞和血小板表面,介导中性粒细胞—内皮细胞、中性粒细胞—血小板等细胞间反应,存在于静止血小板α颗粒及内皮细胞的Weibel-Palade小体中,以可溶性P-选择素(souble P-selectin,sP-selectin)形式出现在血浆中。可溶性P-选择素主要来源于血小板,可以作为血小板激活的标志物。在人血浆里,可溶性P-选择素已经被证实是血小板活化的产物参与血栓形成。编码P-选择素的基因位于人类1号染色体(1q21-24)上一段长50 kb的DNA序列,包含17个外显子。在ECtIM研究中,确定有13个P-选择素的基因多态性:5个分布于启动子远端和8个分布于外显子序列。P-选择素的基因多态性可以影响蛋白的肽序列和调节序列。以往文献报道P-选择素基因位点的多态性,其中包括位于启动子远端与许多转录因子(包括AP-1、c-Fosl3)结合部位的2123 C/G、1817T/C位点和位于第十三外显子的Thr715Pro位点,并且研究表明,可溶性P-选择素水平增高与静脉血栓栓塞事件的风险及P-选择素基因Thr715Pro位点的多态性相关。这些发现表明了P-选择素基因多态性通过调节血浆可溶性P-选择素水平促进血栓栓塞事件的形成。众所周知,心房颤动与血栓栓塞事件的高发生率密切相关,很可能是存在着血栓前或高凝的状态。在血流动力学危险因素中涉及到凝血标志物,某些凝血标志物的基因改变可能增加非辦膜性心房颤动患者发生血栓栓塞事件的风险。最近研究表明,a-纤维蛋白原基因Thr312Ala位点多态性跟心房颤动并发血栓栓塞的发病存在着一定联系。基因的改变在凝血和纤维蛋白溶解途径(凝血危险因素)对心房颤动中高凝状态的发展所起的作用尚需进一步的明确。目前,尚未见P-选择素基因多态性与非辦膜性心房颤动患者血栓事件关联性的研究报告。二、研究目的我们的研究以中国华南地区非辦膜性房颤并发血栓栓塞患者和非辦膜性房颤患者为研究对象,应用现代分子生物学方法,对目前已知的可能涉及房颤血栓栓塞形成的P-选择素基因2123C/G、1817T/C、Thr715Pro位点多态性进行检测,旨在了解中国房颤人群P-选择素基因多态性和可溶性P-选择素水平及其并发血栓栓塞事件关联性的情况,从而在分子水平探索遗传因素在中国房颤人群血栓栓塞事件发病中的作用。三、材料和方法1.以12导联心电图诊断符合心房颤动诊断标准,排除辦膜性心脏病且经CT或MRI诊断为脑卒中或彩色多普勒超声诊断为静脉血栓形成的非辦膜性房颤并发血栓栓塞患者95例(男49例,女46例)和非辦膜性房颤患者106例(男51例,女55例)为对象,测定相关的临床指标,包括:高血压、糖尿病的发生情况,BMI、血脂水平及纤维蛋白原、C反应蛋白。2.采用酶联免疫方法(ELISA)测定入院时血浆中可溶性P-选择素浓度。3.用试剂盒提取静脉血白细胞基因组DNA。P-选择素的基因型用聚合酶链反应及限制性片段长度多态性法(PCR-RFLP)检测,比较P-选择素基因多态性在两组中的差异。在此基础上,分析两组之间其他实验指标结果和上述临床资料。四、结果1、房颤并发血栓栓塞组和对照组临床特征的比较:病例组在总胆固醇(TC)、C反应蛋白水平显著比对照组高(P值分别为0.000和0.009)..两组间平均年龄、性别、体重指数(BMI)、有无吸烟、高血压(HP)、糖尿病、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)均无显著性差异(P＞0.05)。2、P-选择素基因2123C/G位点基因多态性与房颤并发血栓栓塞事件的关系,相对CC基因型而言,GG基因型能显著增加房颤患者血栓事件发生的危险性(P＜0.05),而1817T/C位点各基因型对血栓栓塞事件的发生无显著差异(P＞0.05).3、非辦膜性房颤并发血栓栓塞组可溶性P-选择素血清水平与对照组存在显著性差异(P=0.000)。4、可溶性P-选择素血清水平与总胆固醇、C反应蛋白水平无显著相关。5、可溶性P-选择素血清水平与2123 C/G基因多态性有显著相关性,2123C/G多态性GG基因型者的可溶性P-选择素血清水平显著高于GC+CC基因型(P＜0.05),房颤并发血栓栓塞组和对照组P-选择素基因2123C/G位点的基因型和等位基因分布均有显著性差异(P值分别为0.020和0.015)。6、可溶性P-选择素血清水平与1817T/C基因多态性没有显著性差异(P＞0.05),房颤并发血栓栓塞组和对照组P-选择素基因1817T/C位点的基因型和等位基因分布无显著性差异(P值分别为0.443和0.207)。7、本研究在华南汉族人群中未检测出Thr715Pro基因多态性.五、结论1、P-选择素基因2123 C/G位点的基因多态性与非辦膜性房颤并发血栓栓塞有相关性,GG基因型可能是非辦膜性房颤并发血栓栓塞的易感因素之一。2、P-选择素的基因2123C/G位点的基因多态性可能与血中可溶性P-选择素的浓度或非辦膜性房颤并发血栓栓塞有相关性,然而1817T/C位点无明显关联。3、P-选择素基因Thr715Pro位点多态性不存在于华南汉族人群中。4、本研究揭示了P-选择素的基因多态性可能与血浆中可溶性P-选择素的浓度或血栓栓塞事件有明显的相关性,P-选择素基因可能是通过调节可溶性P-选择素浓度和功能影响非辦膜性房颤患者发生血栓事件的发展进程。更多还原

【Abstract】 BackgroundAssociation between P-selectin gene polymorphisms and soluble P-selectin levels and their relation to thromboembolic events in patients with nonvalvular atrial fibrillation.P-selectin[also known as granule membrane protein-140(GMP-140),platelet activation-dependent granule-external membrane protein(PADGEM) and CD62]is a member of the selectin family of cell adhesion molecules expressed at the surface of activated endothelial cells and platelets,that mediates the interaction of activated endothelial cells or platelets with leukocytes.The protein is localized in the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells,with a soluble form present in the plasma.Levels of plasma P-selectin(soluble P-selectin, sP-selectin) are mainly platelet derived and can be regarded a marker for platelet activatio.In humans,plasma P-selectin levels have therefore been used to substantiate active participation of platelets in thrombotic disease。The P-selectin gene is situated on chromosome 1q21-q24,spans＞50kb and contains 17 exons.In the ECTIM study,thirteen polymorphisms were identified:five in in the distal P-selectin promoter and eight in the exonic sequences.Some polymorphisms of the P-selectin gene have been reported that could affect the peptide sequence of the protein and its regulatory sequences.It is report that the P-selectin gene was identified as being significantly associated with levels of plasma soluble P-selectin(sP-selectin) and thromboembolic events.Several polymorphisms of P-selectin gene has previously been reported including three missense polymorphisms:2123 C/G and 1817 T/C in the distal P-selectin promoter within putative binding sites for a number of transcription factors including AP-1 and c-Fos13,Thr715Pro in exon 13 and plasma levels of P-selectin are especially determined by the P-selectin Thr715Pro polymorphism and high concentrations of soluble P-selectin are associated with risk of venous thromboembolism and the P-selectin Thr715Pro variant.These findings suggest the polymorphisms of P-selectin gene to be involved in the development of thromboembolic events by mediated the plasma soluble P-selectin levels.It is well known that atrial fibrillation is associated with high incidence of thromboembolic events,propably due to a prothrombotic or hypercoagulable state.Some genetic alterations of haemostatic markers in haemostatic risk factors may contribute to the increased risk of thromboembolism in nonvalvular atrial fibrillation patients.Recent studies indicate,the a-fibrinogen Thr312Ala polymorphism contributes to the pathogenesis of thromboembolic complications associated with the presence of atrial fibrillation.The contribution of genetic alterations in factors of the coagulation and fibrinolytic pathways(that is hemostatic risk factors) to the development of hypercoagulation state in atrial fibrillation requires clarification.At present,these is no report that SNPs in P-selectin is associated with thromboembolic events in patients with nonvalvular atrial fibrillation. ObjectivesTo investigate the association between the polymorphism of P-selectin gene in the position of 2123C/G,1817T/C,Thr715Pro and sP-selectin levels and their relation to thromboembolic events in patients with nonvalvular atrial fibrillation(AF) in Han Nationality of Southern China,we used polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) to determine the polymorphism of P-selectin gene in the position of 2123C/G,1817T/C,Thr715Pro.Methods:1.Clinic index of body mass index,blood pressure,plasma lipids,fibrinogen, C-reactive protein were measured in 95 nonvalvular AF patients with thromboembolic events and 106 nonvalvular AF controls without thromboembolic events,who were documented ischemic stroke by MRI and/or CT or venous thromboembolism(VTE) by colour doppler ultrasound.The presence of nonvalvular atrial fibrillation was established on clinical examination at the time of ischemic stroke or venous thromboembolism(VTE) and confirmed on a diagnostic 12-lead ECG and echocardiography.2.Samples were collected when the patients were admitted to hospital.The concentrations of the soluble P-selectin(sP-selectin) was quantified by ELISA3.Genomic DNA in white blood cell from venous blood was abstracted by reagent box.By polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP),we analyzed P-selectin gene polymorphism in patients and controls and compared the distribution of genotype and allele in two group.Based on this,we compared the other result and clinic index including prevalence rate of hypertention and diabetes mellitus,BMI,level of serum cholesterol,triglyceride,high density lipoprotein cholesterol,lowdensity lipoprotein cholesterol,fibrinogenn and C-reactive protein. Results:1.Total cholesterol and C-reactive protein in patient group were significantly higher than those of controls with P value of 0.000 and 0.009 respectively while other clinic features such as age,the component of female,history of cigarette,age,BMI, the morbidity of hypertention and diabetes mellitus,triglyceride,high density lipoprotein cholesterol,low density lipoprotein cholesterol and fibrinogen show no difference between nonvalvular AF patients with thromboembolic events and nonvalvular AF controls without thromboembolic events(P＞0.05).2.The presence of G allele in 2123C/G locus was found to be a greater risk factor in nonvalvular AF subjects with thromboembolic events than in nonvalvular AF controls without thromboembolic events.The odds ratio(OR) of GG were 3.500 (1.401～8.744),when compared with CC genotype,(P＜0.05).however 1817T/C allele show no significant difference(P＞0.05).3.There were statistical differences in levels of sP-selectin between patients and controls(P=0.000).The concentration of the soluble P-selectin is significantly different between patients and controls.4.Soluble P-selectin levels have no correlation with serum cholesterol and C-reactive protein with P value of 0.095and 0.069 respectively.5.The 2123C/G polymorphisms were associated with plasma sP-selectin level and patients with GG genotype tended to present higher level of plasma sP-selectin level than those carrying GC or CC genotype(P＜0.05).Genotype and allele distributions significantly differed in 2123C/G locus with P value of 0.020 and 0.015 respectively between patients and controls.6.Soluble P-selectin levels were not related to 1817T/C allele(P＞0.05) and no significant difference was found in 1817T/C locus genotype and allele distribution frequency with P value of 0.443 and 0.207 respectively between patients and controls. 7.The Thr715Pro polymorphism was not found in Han Nationality of Southern China in this study.Conclusions1.The 2123C/G polymorphism of P-selectin gene was associated with thromboembolic events in patients with nonvalvular atrial fibrillation,the genotype of GG may be a genetic risk factor for the thromboembolic process in patients with nonvalvular AF.2.The 2123C/G polymorphism of P-selectin gene was associated with soluble P-selectin levels or thromboembolic events in patients with nonvalvular atrial fibrillation,however 1817T/C locus polymorphism was not associated.3.Thr715Pro locus SNP does not exist in Han Nationality of Southern China.4.This study revealed a relationship between P-selectin gene polymorphisms and serum sP-selectin levels or thromboembolic events.The P-selectin gene affect the development of the thromboembolic process in patients with nonvalvular AF by mediating the concentration and function of soluble P-selectin.更多还原