蛋白质Z水平及其基因多态性与急性缺血性脑卒中的相关性研究
发布时间：2019-07-01 15:06
【摘要】：研究背景： 随着我国逐渐跨入老龄化社会，老年人口越来越多，急性血栓性脑血管疾病的发病率越来越高，已成为我国老年人最常见的死亡原因之一。血栓形成的病理过程是复杂的，其作用机制包括血管因素、凝血、抗凝以及纤溶功能异常等。探讨血栓形成的发病病理过程，对诊断和治疗临床血栓性疾病具有重要的指导意义。蛋白质Z（proteinZ，PZ）是维生素K依赖的血浆蛋白，为蛋白质Z依赖性蛋白酶抑制剂（ProteinZ-dependent protease inhibitor,ZPI）抑制凝血因子Xa的辅因子，PZ能提高ZPI抑制凝血因子Xa的活性，在出血及血栓性疾病中发挥双向调节作用。最近国外有少量的文献报道，血浆PZ水平及其基因多态性与急性缺血性脑卒中的患病风险相关，但研究结果不一。本研究首次在中国汉族人群中分析血浆PZ水平及其基因多态性与急性缺血性脑卒中(Acute ischemic stroke,AIS）的相关性，并探讨其可能的作用机制，为临床上的预测、诊断与治疗提供一定理论依据。 目的： 1.探讨血浆PZ水平在急性缺血性脑卒中发生的意义。 2.探讨PZ基因多态性与急性缺血性脑卒中的相关性。 3.揭示血浆PZ水平与其启动子A-13G和内含子F中G79A两种基因多态之间的相关性。 4.填补中国人群在PZ水平及基因多态性与急性缺血性脑卒中相关性研究中的空白。 方法： 第一部分测定急性缺血性脑卒中患者PZ血浆变化 完成收集AIS患者、健康对照组的血液样品后，用ELISA法检测各组血浆PZ水平。实验分2组：急性缺血性脑卒中患者急性期组（初发组和再发组）、正常对照组。严格按照PZ ELISA试剂盒说明书完成实验过程。完成统计分析，，探讨AIS患者血浆PZ水平变化。 第二部分测定急性缺血性脑卒中患者PZ基因多态性 实验分2组：AIS组、正常对照组。每个血液样品收集完成后，用盐析法提取外周血基因组DNA，对基因组DNA进行聚合酶链反应（Polymerase Chain Reaction, PCR）扩增，并利用限制性内切酶片段长度多态性方法结合基因测序技术，检测PZ启动子A-13G及其内含子F中G79A两种基因多态性。 第三部分分析PZ血浆水平与其基因多态性的相关性 PZ血浆水平比较采用t检验统计；多态性位点基因型和等位基因频率比较采用卡方检验；计算各组中等位基因和基因型分布频率采用Hardy-Weinberg平衡检验；PZ血浆水平与其基因多态性的相关性采用Spearman秩相关系数表示；PZ基因型和等位基因与AIS风险预测采用比值比(OR)及其95%的可信区间(CI)来表示。 结果： 第一部分AIS患者血浆PZ水平变化 1. AIS组的血浆PZ水平显著低于对照组（P0.01）。 2. AIS再发组PZ水平明显低于初发组(P0.01)。 第二部分PZ启动子A-13G及内含子G79A基因多态性分布 1. AIS组PZ基因内含子F中G79A位点G等位基因频率显著高于对照组（P0.05）。 2. AIS组PZ基因内含子F中G79A位点A等位基因频率明显低于对照组(P0.05)。 3. AIS组G79A位点与对照组的基因型AA型（29.0%vs34.0%）、AG型（50.1%vs54.1%）、GG型20.9%vs11.9%），差异有统计学意义（P=0.05）。 4. AIS组A-13G位点等位基因频率及基因型（AG型、GG型、AA型）与对照组没有明显差异（P＞0.05）。 5.对照组PZ启动子A-13G的G等位基因频率显著高于欧洲白种人群（60.7%vs13.1%），PZ内含子F中G79A的A等位基因频率显著高于欧洲白种人群（61.1%vs40.7%）。 第三部分PZ血浆水平与其基因多态性的相关性 1. AIS组PZ内含子F中G79A中AA基因型的PZ水平明显低于对照组（P0.05）。 2. AIS组PZ内含子F中G79A中GG基因型的PZ水平明显高于对照组（P0.05）。 3. AIS组PZ启动子A-13G各基因型的PZ水平与对照组无明显差异（P＞0.05）。 4. AIS与吸烟、高血压、高血脂及糖尿病这些危险因子明显相关（P0.01）。 结论： 1.血浆PZ水平与AIS相关,表明PZ的缺乏可能是急性缺血性脑卒中疾病存在的一个危险因素。 2. PZ启动子A-13G位点A等位基因频率及基因型与AIS无相关性，显示PZ基因多态性A-13G位点可能不参与AIS疾病的发生、发展。 3. PZ基因G79A位点AA基因型和A等位基因的频率与AIS呈负相关，表明AIS患者PZ基因内含子G79A位点存在G→A突变。 4. PZ基因G79A位点基因型AA、GA、GG中的AA基因型的血浆PZ水平与AIS负相关，提示AA基因型的PZ水平可能在血栓性疾病中通过表达下调方式，从而降低血栓性疾病发生的风险，反映AA基因型可作为AIS患者的保护因子。 5. PZ基因突变存在种族差异，中国珠三角地区汉族人群突变率显著高于欧洲白种人群。 6. AIS与吸烟、高血压、高血脂及糖尿病这些危险因子明显相关,改变这些危险因子，能够降低AIS的发病率和再发率。
[Abstract]:Study Background: With the development of the aging society, the more and more old people, the incidence of acute thrombotic cerebrovascular diseases is higher and higher, which has become the most common cause of death in the old people in our country. I. The pathological process of thrombosis is complex, and its mechanism of action includes vascular factors, coagulation, anticoagulation, and abnormal fibrinolysis. And so on. To study the pathological process of thrombosis, it is of great guide to the diagnosis and treatment of clinical thrombotic diseases. The protein Z (PZ) is the plasma protein which is dependent on the vitamin K. The protein Z-dependent protease inhibitor (ZPI) can inhibit the factor Xa, and the PZ can improve the activity of the blood coagulation factor Xa by the ZPI, and play a two-way regulation in the bleeding and the thrombotic diseases. In recent years, a small number of literatures have reported that the plasma PZ level and its gene polymorphism are related to the risk of acute ischemic stroke, but the results of the study do not First, the relationship between the plasma PZ level and its gene polymorphism and acute ischemic stroke (AIS) in the Chinese Han population was analyzed for the first time in the Chinese Han population, and its possible mechanism of action was discussed, which provided some theoretical basis for the clinical prediction, diagnosis and treatment. It was reported. Objective:1. To study the plasma PZ level in acute ischemic stroke the significance of the occurrence of PZ gene polymorphism and acute ischemic 3. The relationship between plasma PZ level and its promoter A-13G and G79A in intron F was revealed. 4. To fill the correlation between polymorphism and PZ level and gene polymorphism and acute ischemic brain in Chinese population. stroke Blank in the closed study. Method: first part determination the blood samples of the AIS patients and the healthy control group are collected after the PZ plasma changes of the patients with acute ischemic stroke are completed, Plasma PZ levels in each group were detected by ELISA. Period group (primary and secondary groups), normal control group. strictly according to PZ The test procedure was completed by the ELISA kit specification. The statistical score was completed. A study on the changes of plasma PZ level in patients with AIS. Determination of PZ gene in patients with acute ischemic stroke The polymorphism was divided into two groups: the AIS group and the normal control group. After the collection of each blood sample, the genomic DNA of the peripheral blood was extracted by the salting-out method, and the genomic DNA was amplified by polymerase chain reaction (PCR) and the restriction was used. the invention relates to a method for detecting the length polymorphism of an endoenzyme fragment and a gene sequencing technology to detect a PZ promoter A- Genetic polymorphism of G79A in 13G and its intron F. In the third part, the relationship between the plasma level of PZ and its gene polymorphism was analyzed by t-test. The genotype of the polymorphism and the frequency of the allele were compared with the chi-square test, and the alleles and genes in each group were calculated. The frequency of distribution is Hardy-Weinberg equilibrium test; the correlation between the PZ plasma level and its gene polymorphism is expressed by the Spearman rank correlation coefficient; the PZ genotype and the allele and the AIS risk prediction ratio (OR) and its 95% confidence interval (C I). Results: The plasma PZ level in the first part of the AIS patient The plasma PZ level in the AIS group was significantly lower than that in the control group (P0 (01).2. The level of PZ in the AIS regenerator group was significantly lower than that of the primary group (P0 .01). The second part of the PZ promoter A-13G and the intron G79A gene polymorphism distribution 1.AIS The G allele frequency of the G79A locus in the intron F of the group PZ gene was significantly higher than that in the control group (P0.05). The allele frequency of the G79A locus in the intron F of the PZ gene in the IS group was significantly lower than that in the control group (P0.05). s54.1%), GG (20.9% vs11.9%), statistical significance (P = 0.05).4. AIS group A-13G In the control group, the G allele frequency of the PZ promoter A-13G was significantly higher in the control group than in the European Caucasians (60.7% vs1.3%). ), the A allele frequency of the G79A in the PZ intron F significantly higher than that in the European Caucasians (61.1% vs40.7%). The third part of the PZ plasma level and its gene polymorphism The PZ level of the AA genotype in the PZ intron F of the AIS group was significantly lower than that of the control group ( 2. The PZ level of the GG genotype in the PZ intron F of the AIS group was significantly higher than that of the control group. Group (P0.05).3. The PZ level of the PZ promoter A-13G in the AIS group and the pair Group None 4. AIS was associated with the risk factors of smoking, hypertension, hyperlipidemia and diabetes. Conclusion:1. The plasma PZ level is associated with AIS, indicating that the deficiency of PZ may be a risk factor for acute ischemic stroke. The allele frequency and genotype of the A-13G locus of the mover A-13G are not related to the AIS, and the polymorphism of the polymorphism A-13G of the PZ gene may not be involved in the occurrence and development of the AIS disease. 3. The frequency of the G79A site of the PZ gene and the frequency of the A allele were negatively correlated with the AIS, indicating that there was a G-A mutation in the G79A site of the PZ gene in the AIS patients. The plasma PZ level of the AA genotype was negatively correlated with the AIS. It is suggested that the PZ level of the AA genotype may be down-regulated by the expression down-regulation in the thrombotic diseases, thereby reducing the risk of thrombotic diseases. The risk of birth is that the AA genotype can be used as the protective factor of the AIS patients.
【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3
[Abstract]:Study Background: With the development of the aging society, the more and more old people, the incidence of acute thrombotic cerebrovascular diseases is higher and higher, which has become the most common cause of death in the old people in our country. I. The pathological process of thrombosis is complex, and its mechanism of action includes vascular factors, coagulation, anticoagulation, and abnormal fibrinolysis. And so on. To study the pathological process of thrombosis, it is of great guide to the diagnosis and treatment of clinical thrombotic diseases. The protein Z (PZ) is the plasma protein which is dependent on the vitamin K. The protein Z-dependent protease inhibitor (ZPI) can inhibit the factor Xa, and the PZ can improve the activity of the blood coagulation factor Xa by the ZPI, and play a two-way regulation in the bleeding and the thrombotic diseases. In recent years, a small number of literatures have reported that the plasma PZ level and its gene polymorphism are related to the risk of acute ischemic stroke, but the results of the study do not First, the relationship between the plasma PZ level and its gene polymorphism and acute ischemic stroke (AIS) in the Chinese Han population was analyzed for the first time in the Chinese Han population, and its possible mechanism of action was discussed, which provided some theoretical basis for the clinical prediction, diagnosis and treatment. It was reported. Objective:1. To study the plasma PZ level in acute ischemic stroke the significance of the occurrence of PZ gene polymorphism and acute ischemic 3. The relationship between plasma PZ level and its promoter A-13G and G79A in intron F was revealed. 4. To fill the correlation between polymorphism and PZ level and gene polymorphism and acute ischemic brain in Chinese population. stroke Blank in the closed study. Method: first part determination the blood samples of the AIS patients and the healthy control group are collected after the PZ plasma changes of the patients with acute ischemic stroke are completed, Plasma PZ levels in each group were detected by ELISA. Period group (primary and secondary groups), normal control group. strictly according to PZ The test procedure was completed by the ELISA kit specification. The statistical score was completed. A study on the changes of plasma PZ level in patients with AIS. Determination of PZ gene in patients with acute ischemic stroke The polymorphism was divided into two groups: the AIS group and the normal control group. After the collection of each blood sample, the genomic DNA of the peripheral blood was extracted by the salting-out method, and the genomic DNA was amplified by polymerase chain reaction (PCR) and the restriction was used. the invention relates to a method for detecting the length polymorphism of an endoenzyme fragment and a gene sequencing technology to detect a PZ promoter A- Genetic polymorphism of G79A in 13G and its intron F. In the third part, the relationship between the plasma level of PZ and its gene polymorphism was analyzed by t-test. The genotype of the polymorphism and the frequency of the allele were compared with the chi-square test, and the alleles and genes in each group were calculated. The frequency of distribution is Hardy-Weinberg equilibrium test; the correlation between the PZ plasma level and its gene polymorphism is expressed by the Spearman rank correlation coefficient; the PZ genotype and the allele and the AIS risk prediction ratio (OR) and its 95% confidence interval (C I). Results: The plasma PZ level in the first part of the AIS patient The plasma PZ level in the AIS group was significantly lower than that in the control group (P0 (01).2. The level of PZ in the AIS regenerator group was significantly lower than that of the primary group (P0 .01). The second part of the PZ promoter A-13G and the intron G79A gene polymorphism distribution 1.AIS The G allele frequency of the G79A locus in the intron F of the group PZ gene was significantly higher than that in the control group (P0.05). The allele frequency of the G79A locus in the intron F of the PZ gene in the IS group was significantly lower than that in the control group (P0.05). s54.1%), GG (20.9% vs11.9%), statistical significance (P = 0.05).4. AIS group A-13G In the control group, the G allele frequency of the PZ promoter A-13G was significantly higher in the control group than in the European Caucasians (60.7% vs1.3%). ), the A allele frequency of the G79A in the PZ intron F significantly higher than that in the European Caucasians (61.1% vs40.7%). The third part of the PZ plasma level and its gene polymorphism The PZ level of the AA genotype in the PZ intron F of the AIS group was significantly lower than that of the control group ( 2. The PZ level of the GG genotype in the PZ intron F of the AIS group was significantly higher than that of the control group. Group (P0.05).3. The PZ level of the PZ promoter A-13G in the AIS group and the pair Group None 4. AIS was associated with the risk factors of smoking, hypertension, hyperlipidemia and diabetes. Conclusion:1. The plasma PZ level is associated with AIS, indicating that the deficiency of PZ may be a risk factor for acute ischemic stroke. The allele frequency and genotype of the A-13G locus of the mover A-13G are not related to the AIS, and the polymorphism of the polymorphism A-13G of the PZ gene may not be involved in the occurrence and development of the AIS disease. 3. The frequency of the G79A site of the PZ gene and the frequency of the A allele were negatively correlated with the AIS, indicating that there was a G-A mutation in the G79A site of the PZ gene in the AIS patients. The plasma PZ level of the AA genotype was negatively correlated with the AIS. It is suggested that the PZ level of the AA genotype may be down-regulated by the expression down-regulation in the thrombotic diseases, thereby reducing the risk of thrombotic diseases. The risk of birth is that the AA genotype can be used as the protective factor of the AIS patients.
【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3
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