蛋白質(zhì)Z水平及其基因多態(tài)性與急性缺血性腦卒中的相關(guān)性研究
發(fā)布時(shí)間：2019-07-01 15:06
【摘要】：研究背景： 隨著我國(guó)逐漸跨入老齡化社會(huì)，老年人口越來(lái)越多，急性血栓性腦血管疾病的發(fā)病率越來(lái)越高，已成為我國(guó)老年人最常見的死亡原因之一。血栓形成的病理過(guò)程是復(fù)雜的，其作用機(jī)制包括血管因素、凝血、抗凝以及纖溶功能異常等。探討血栓形成的發(fā)病病理過(guò)程，對(duì)診斷和治療臨床血栓性疾病具有重要的指導(dǎo)意義。蛋白質(zhì)Z（proteinZ，PZ）是維生素K依賴的血漿蛋白，為蛋白質(zhì)Z依賴性蛋白酶抑制劑（ProteinZ-dependent protease inhibitor,ZPI）抑制凝血因子Xa的輔因子，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發(fā)揮雙向調(diào)節(jié)作用。最近國(guó)外有少量的文獻(xiàn)報(bào)道，血漿PZ水平及其基因多態(tài)性與急性缺血性腦卒中的患病風(fēng)險(xiǎn)相關(guān)，但研究結(jié)果不一。本研究首次在中國(guó)漢族人群中分析血漿PZ水平及其基因多態(tài)性與急性缺血性腦卒中(Acute ischemic stroke,AIS）的相關(guān)性，并探討其可能的作用機(jī)制，為臨床上的預(yù)測(cè)、診斷與治療提供一定理論依據(jù)。 目的： 1.探討血漿PZ水平在急性缺血性腦卒中發(fā)生的意義。 2.探討PZ基因多態(tài)性與急性缺血性腦卒中的相關(guān)性。 3.揭示血漿PZ水平與其啟動(dòng)子A-13G和內(nèi)含子F中G79A兩種基因多態(tài)之間的相關(guān)性。 4.填補(bǔ)中國(guó)人群在PZ水平及基因多態(tài)性與急性缺血性腦卒中相關(guān)性研究中的空白。 方法： 第一部分測(cè)定急性缺血性腦卒中患者PZ血漿變化 完成收集AIS患者、健康對(duì)照組的血液樣品后，用ELISA法檢測(cè)各組血漿PZ水平。實(shí)驗(yàn)分2組：急性缺血性腦卒中患者急性期組（初發(fā)組和再發(fā)組）、正常對(duì)照組。嚴(yán)格按照PZ ELISA試劑盒說(shuō)明書完成實(shí)驗(yàn)過(guò)程。完成統(tǒng)計(jì)分析，，探討AIS患者血漿PZ水平變化。 第二部分測(cè)定急性缺血性腦卒中患者PZ基因多態(tài)性 實(shí)驗(yàn)分2組：AIS組、正常對(duì)照組。每個(gè)血液樣品收集完成后，用鹽析法提取外周血基因組DNA，對(duì)基因組DNA進(jìn)行聚合酶鏈反應(yīng)（Polymerase Chain Reaction, PCR）擴(kuò)增，并利用限制性內(nèi)切酶片段長(zhǎng)度多態(tài)性方法結(jié)合基因測(cè)序技術(shù)，檢測(cè)PZ啟動(dòng)子A-13G及其內(nèi)含子F中G79A兩種基因多態(tài)性。 第三部分分析PZ血漿水平與其基因多態(tài)性的相關(guān)性 PZ血漿水平比較采用t檢驗(yàn)統(tǒng)計(jì)；多態(tài)性位點(diǎn)基因型和等位基因頻率比較采用卡方檢驗(yàn)；計(jì)算各組中等位基因和基因型分布頻率采用Hardy-Weinberg平衡檢驗(yàn)；PZ血漿水平與其基因多態(tài)性的相關(guān)性采用Spearman秩相關(guān)系數(shù)表示；PZ基因型和等位基因與AIS風(fēng)險(xiǎn)預(yù)測(cè)采用比值比(OR)及其95%的可信區(qū)間(CI)來(lái)表示。 結(jié)果： 第一部分AIS患者血漿PZ水平變化 1. AIS組的血漿PZ水平顯著低于對(duì)照組（P0.01）。 2. AIS再發(fā)組PZ水平明顯低于初發(fā)組(P0.01)。 第二部分PZ啟動(dòng)子A-13G及內(nèi)含子G79A基因多態(tài)性分布 1. AIS組PZ基因內(nèi)含子F中G79A位點(diǎn)G等位基因頻率顯著高于對(duì)照組（P0.05）。 2. AIS組PZ基因內(nèi)含子F中G79A位點(diǎn)A等位基因頻率明顯低于對(duì)照組(P0.05)。 3. AIS組G79A位點(diǎn)與對(duì)照組的基因型AA型（29.0%vs34.0%）、AG型（50.1%vs54.1%）、GG型20.9%vs11.9%），差異有統(tǒng)計(jì)學(xué)意義（P=0.05）。 4. AIS組A-13G位點(diǎn)等位基因頻率及基因型（AG型、GG型、AA型）與對(duì)照組沒有明顯差異（P＞0.05）。 5.對(duì)照組PZ啟動(dòng)子A-13G的G等位基因頻率顯著高于歐洲白種人群（60.7%vs13.1%），PZ內(nèi)含子F中G79A的A等位基因頻率顯著高于歐洲白種人群（61.1%vs40.7%）。 第三部分PZ血漿水平與其基因多態(tài)性的相關(guān)性 1. AIS組PZ內(nèi)含子F中G79A中AA基因型的PZ水平明顯低于對(duì)照組（P0.05）。 2. AIS組PZ內(nèi)含子F中G79A中GG基因型的PZ水平明顯高于對(duì)照組（P0.05）。 3. AIS組PZ啟動(dòng)子A-13G各基因型的PZ水平與對(duì)照組無(wú)明顯差異（P＞0.05）。 4. AIS與吸煙、高血壓、高血脂及糖尿病這些危險(xiǎn)因子明顯相關(guān)（P0.01）。 結(jié)論： 1.血漿PZ水平與AIS相關(guān),表明PZ的缺乏可能是急性缺血性腦卒中疾病存在的一個(gè)危險(xiǎn)因素。 2. PZ啟動(dòng)子A-13G位點(diǎn)A等位基因頻率及基因型與AIS無(wú)相關(guān)性，顯示PZ基因多態(tài)性A-13G位點(diǎn)可能不參與AIS疾病的發(fā)生、發(fā)展。 3. PZ基因G79A位點(diǎn)AA基因型和A等位基因的頻率與AIS呈負(fù)相關(guān)，表明AIS患者PZ基因內(nèi)含子G79A位點(diǎn)存在G→A突變。 4. PZ基因G79A位點(diǎn)基因型AA、GA、GG中的AA基因型的血漿PZ水平與AIS負(fù)相關(guān)，提示AA基因型的PZ水平可能在血栓性疾病中通過(guò)表達(dá)下調(diào)方式，從而降低血栓性疾病發(fā)生的風(fēng)險(xiǎn)，反映AA基因型可作為AIS患者的保護(hù)因子。 5. PZ基因突變存在種族差異，中國(guó)珠三角地區(qū)漢族人群突變率顯著高于歐洲白種人群。 6. AIS與吸煙、高血壓、高血脂及糖尿病這些危險(xiǎn)因子明顯相關(guān),改變這些危險(xiǎn)因子，能夠降低AIS的發(fā)病率和再發(fā)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.
【學(xué)位授予單位】：廣東藥學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：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.
【學(xué)位授予單位】：廣東藥學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743.3
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