KDR基因rs2305948、rs2239702多态性与缺血性中风、冠心病痰瘀证凝血功能的关联性研究
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摘要
目的探讨激酶插入结构域受体(KDR)基因多态性与缺血性中风(IS)痰瘀证、冠心病(CAD)痰瘀证及凝血功能的关联性。方法共纳入缺血性中风痰瘀证患者550例、冠心病痰瘀证患者550例、正常对照人群550例,分为3组。采用MassARRAY SNP基因分型实验技术进行基因分型检测,使用实时荧光定量PCR(RT-PCR)方法检测外周血KDR基因mRNA表达水平,通过凝固法检测血浆中凝血功能指标。采用PLINK软件进行遗传关联分析,采用拟和优度χ~2检验进行哈温平衡(HWE)分析。基因型频率分布的组间比较,采用χ~2检验。应用多因素非条件Logistic回归模型对各遗传模型(加性模型、显性模型、隐性模型)与疾病的关联进行分析,采用线性回归分析各位点多态性与凝血标志物水平的关联。结果 IS痰瘀证患者的KDR基因mRNA表达水平显著低于对照组(P<0.001),而CAD痰瘀证患者的KDR基因mRNA表达水平显著高于对照组(P=0.007)。KDR基因rs2305948、rs2239702多态性与IS痰瘀证、CAD痰瘀证发生风险的关联差异无统计学意义(均为P>0.05)。KDR基因rs2305948多态性与IS痰瘀证患者的D二聚体(D-D)、凝血酶原时间活动度(PTA)的相关性具有统计学意义(P<0.05),KDR基因rs2239702多态性与IS痰瘀证患者的活化部分凝血酶原时间(APTT)显著相关(P<0.05)。KDR基因rs2305948多态性与CAD痰瘀证患者APTT水平的相关性具有统计学意义(P<0.05),rs2239702多态性与CAD痰瘀证患者凝血酶时间(TT)水平显著相关(P<0.05)。结论 KDR基因表达水平可能影响IS痰瘀证、CAD痰瘀证的发生,且KDR基因rs2305948、rs2239702遗传多态性可能影响IS痰瘀证、CAD痰瘀证的凝血功能。
Objective To investigate the correlation between KDR gene polymorphism and phlegm-stasis syndrome in ischemic stroke(IS), coronary heart disease(CAD) and coagulation function. Methods A total of 550 cases of ischemic stroke with phlegm and blood stasis syndrome, 550 cases of coronary heart disease with phlegm and blood stasis syndrome, and 550 subjects of normal control were included. Genotyping was performed using MassARRAy SNP genotyping assay technology, and the mRNA expression level of KDR gene in peripheral blood was detected with real-time fluorescence quantitative PCR(RT-PCR) method, and the coagulation function index in plasma was detected with coagulation method. PLINK software was used for genetic association analysis, and χ~2 test was used for ha-temperature equilibrium(HWE) analysis. Genotype frequency distribution was compared between groups by χ~2 test. Multivariate unconditioned Logistic regression model was used to analyze the correlation between genetic models(additive model, dominant model and recessive model) and disease, and linear regression was used to analyze the correlation between polymorphism at each point and the level of coagulation markers. Results The expression level of KDR gene mRNA in IS patients with phlegm and blood stasis syndrome was significantly lower than that in the control group(P<0.001), while the expression level of KDR gene mRNA in CAD patients with phlegm and blood stasis syndrome was significantly higher than that in the control group(P =0.007). KDR gene rs2305948, rs2239702 polymorphism and IS phlegm and blood stasis syndrome, CAD phlegm and blood stasis syndrome risk was not statistically significant(P>0.05). KDR gene rs2305948 polymorphism was significantly correlated with D-dimer(D-D) and prothrombin time activity(PTA) in IS patients with phlegm and blood stasis syndrome(P<0.05). KDR gene rs2239702 polymorphism was significantly correlated with activated partial prothrombin time(APTT) in IS patients with phlegm and blood stasis syndrome(P<0.05). KDR gene rs2305948 polymorphism was significantly correlated with APTT level in CAD patients with phlegm and blood stasis syndrome(P<0.05), and rs2239702 polymorphism was significantly correlated with thrombin time(TT) level in CAD patients with phlegm and blood stasis syndrome(P<0.05). Conclusion The expression of KDR gene may be involved in the occurrence of IS phlegm-stasis syndrome and CAD phlegm-stasis syndrome, and the genetic polymorphism of KDR gene rs2305948 and rs2239702 may affect the coagulation function of IS phlegm-stasis syndrome and CAD phlegm-stasis syndrome.
Objective To investigate the correlation between KDR gene polymorphism and phlegm-stasis syndrome in ischemic stroke(IS), coronary heart disease(CAD) and coagulation function. Methods A total of 550 cases of ischemic stroke with phlegm and blood stasis syndrome, 550 cases of coronary heart disease with phlegm and blood stasis syndrome, and 550 subjects of normal control were included. Genotyping was performed using MassARRAy SNP genotyping assay technology, and the mRNA expression level of KDR gene in peripheral blood was detected with real-time fluorescence quantitative PCR(RT-PCR) method, and the coagulation function index in plasma was detected with coagulation method. PLINK software was used for genetic association analysis, and χ~2 test was used for ha-temperature equilibrium(HWE) analysis. Genotype frequency distribution was compared between groups by χ~2 test. Multivariate unconditioned Logistic regression model was used to analyze the correlation between genetic models(additive model, dominant model and recessive model) and disease, and linear regression was used to analyze the correlation between polymorphism at each point and the level of coagulation markers. Results The expression level of KDR gene mRNA in IS patients with phlegm and blood stasis syndrome was significantly lower than that in the control group(P<0.001), while the expression level of KDR gene mRNA in CAD patients with phlegm and blood stasis syndrome was significantly higher than that in the control group(P =0.007). KDR gene rs2305948, rs2239702 polymorphism and IS phlegm and blood stasis syndrome, CAD phlegm and blood stasis syndrome risk was not statistically significant(P>0.05). KDR gene rs2305948 polymorphism was significantly correlated with D-dimer(D-D) and prothrombin time activity(PTA) in IS patients with phlegm and blood stasis syndrome(P<0.05). KDR gene rs2239702 polymorphism was significantly correlated with activated partial prothrombin time(APTT) in IS patients with phlegm and blood stasis syndrome(P<0.05). KDR gene rs2305948 polymorphism was significantly correlated with APTT level in CAD patients with phlegm and blood stasis syndrome(P<0.05), and rs2239702 polymorphism was significantly correlated with thrombin time(TT) level in CAD patients with phlegm and blood stasis syndrome(P<0.05). Conclusion The expression of KDR gene may be involved in the occurrence of IS phlegm-stasis syndrome and CAD phlegm-stasis syndrome, and the genetic polymorphism of KDR gene rs2305948 and rs2239702 may affect the coagulation function of IS phlegm-stasis syndrome and CAD phlegm-stasis syndrome.
引文
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[15] 张红珍.浅谈动脉粥样硬化与虚瘀痰浊[J].新中医,2012,44(8):173-175.Zhang HZ.Discussion on atherosclerosis and deficien cy and stagnation of phlegm[J].Journal of New Chi nese Medicine,2012,44(8):173-175.
[16] Holmes K,Roberts OL,Thomas AM,et al.Vascular en dothelial growth factor receptor-2:structure,function,intracellular signaling and therapeutic inhibition[J].Cel lular Signaling,2007,19(10):2003-2012.
[17] 刘利妍,刘向群,陈效安,等.大鼠动脉粥样硬化形成过程中TAFI水平变化及作用机制[J].山东大学学报(医学版),2007,45(8):787-791.Liu LY,liu XQ,Chen XA,et al.TAFI level and its mech anism in the process of atherosclerosis in rats[J].Jour nal of Shandong University (Health Sciences),2007,45(8):787-791.
[18] Guo M,Sahni SK,Sahni A,et al.Fibrinogen regulates the expression of inflammatory chemokines through NF-kappaB activation of endothelial cells[J].Thrombo sis & Haemostasis,2004,92(4):858-866.
[19] 左丽,周鹏.血浆LOX-1与人外周血单核细胞LOX-1 mRNA表达水平与冠脉病变程度的相关性研究[J].川北医学院学报,2013,33(6):807-811.Zuo L,Zhou P.The relationship between angiographic coronary artery lesions and the expression of serum lox-1 and lox-1 mRNA on circulating monocytes[J].Journal of North Sichuan Medical College,2013,33(6):807-811.
[20] 王伟,黄静妍,谭金晶,等.Toll样受体5基因遗传多态性与中风风痰瘀阻证显著关联的探讨[J].辽宁中医杂志,2016,43(12):2495-2497.Wang W,Huang JY,Tan JJ,et al.Genetic polymor phism of toll-like receptor 5 gene significantly associat ed with stroke wind-phlegm-stasis syndrome[J].Liaon ing Journal of Traditional Chinese Medicine,2016,43(12):2495-2497.
[21] 陈琪.β纤维蛋白原-455G/A基因多态性与冠心病的关系[J].中国动脉硬化杂志,2013,21(10):931-934.Chen Q.Association of β-455G/A polymorphism with coronary heart disease[J].Chinese Journal of Atherosclerosis,2013,21(10):931-934.
[22] 何娴,叶晓冰,陈家钖,等.脑卒中患者载脂蛋白E基因多态性与血小板水平相关性研究[J].黑龙江医药,2018,31(1):118-120.He X,Ye XB,Chen JD,et al.Correlation between apoli poprotein E gene polymorphism and platelet level in stroke patients [J].Heilongjiang pharmaceutical,2018,31(1):118-120.
[23] 周明权.冠心病患者凝血四项及纤维蛋白原多态性分析[J].中国实用医药,2012,7(5):70-71.Zhou MQ.Analysis of four clotting items and fibrinogen polymorphism in patients with coronary heart disease[J].Chinese Journal of Practical Medicine,2012,7(5):70-71.
[24] 袁蓉,王阶,郭丽丽.冠心病痰瘀互结证的近代研究及中医治疗进展[J].中国中药杂志,2016,41(1):35-37.Yuan R,Wang T,Guo LL.Advances in modern research and TCM treatment of phlegm and blood stasis syn drome of coronary heart disease[J].Chinese Journal of Traditional Chinese Medicine,2016,41 (1):35-37.
[25] 任建勋,刘建勋,林成仁,等.冠心病不稳定型心绞痛痰瘀互结证临床生化指标研究[J].中医杂志,2012,53(8):665-668.Ren JX,Liu JX,Lin CR,et al.The biochemical indicators in coronary heart disease patients with unstable angina pectoris[J].Journal of Traditional Chinese Medicine,2012,53(8):665-668.
[2] Shalaby F,Rossant J,Yamaguchi TP,et al.Failure of blood-island formation and vasculogenesis in Flk-1-defi cient mice[J].Nature,1995,376(6535):62.
[3] Gerber HP,Dixit V,Ferrara N.Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells[J].Journal of Biological Chemistry,1998,273(21):13313-13316.
[4] Gerber HP,Mcmurtrey A,Kowalski J,et al.Vascular en dothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway:requirement for Flk-1/KDR activation[J].Journal of Biological Chemistry,1998,273(46):30336-30343.
[5] 张伟丽,惠汝太.VEGF及其受体在动脉粥样硬化中的作用[J].中国分子心脏病学杂志,2005,5(3):568-573.Zhang WL,Hui RT.Role of vascular endothelial growth factor and its receptor in atherosclerosis[J].Molecular Cardiology of China,2005,5(3):568-573.
[6] Oh SH,Min KT,Jeon YJ,et al.Association between ki nase insert domain-containing receptor gene polymor phism and haplotypes and ischemic stroke[J].Journal of the Neurological Sciences,2011,308(1-2):62-66.
[7] Wang YB,Zheng Y,Zhang WL,et al.Polymorphisms of KDR gene are associated with coronary heart disease[J].Journal of the American College of Cardiology,2007,50(8):760-767.
[8] 王新德.各类脑血管疾病诊断要点[J].中华神经科杂志,1996,29(6):379-380.Wang XD.Diagnosis of various cerebrovascular diseases[J].Chinese journal of neurology,1996,29(6):379 -380.
[9] 国家中医药管理局脑病急症协作组.中风病诊断与疗效评定标准(试行)[J].北京中医药大学学报,1996,19(1):55-56.Brain Disease Emergency Collaborative Group,SATCM.Criteria for the Diagnosis and Therapeutic Evaluation of Stroke(tentative)[J].Journal of Beijing University of Traditional Chinese Medicine,1996,19(1):55-56.
[10] 徐济民.缺血性心脏病诊断的命名及标准——国际心脏病学会和协会/世界卫生组织临床命名标准化专题组的联合报告[J].国外医学.心血管疾病分册,1979,6(6):365-366.Xu JM.nomenclature and criteria for the diagnosis of ischemic heart disease:a joint report of the international society of cardiology and the international association of cardiology/who task force on standardization of clini cal nomenclature [J].Foreign Medsci:Cardioangiopa thy,1979,6(6):365-366.
[11] 中国中西医结合学会心血管学会.冠心病中医辨证标准[J].中西医结合杂志,1991,11(5):257-257.Cardiovascular Society of China Association of Inte grative Medicine.Standard of TCM syndrome differenti ation for coronary heart disease[J].Journal of inte g rated Chinese and western medicine,1991,11(5):257 -257.
[12] 李小兵,方永奇,洪永敦,等.心脑血管病痰证宏观辨证的计量化研究[J].中国中医基础医学杂志,2000,6(5):44-47.Li XB,Fang YQ,Hong YD,et al.Quantitative study on macroscopic differentiation of phlegm syndrome in car diovascular and cerebrovascular diseases [J].Chinese journal of basic medicine of traditional Chinese medi cine,2000,6(5):44-47.
[13] 王阶,陈可冀,翁维良,等.血瘀证诊断标准的研究[J].中国中西医结合杂志,1988,8(10):585-587.Wang J,Chen KJ,Weng WL,et al.Study on diagnostic criteria of blood stasis syndrome [J].Chinese Journal of Integrated Traditional Chinese and Western medicine,1988,8(10):585-587.
[14] 王佳馨,杨关林.中风与胸痹病机特点诠释[J].实用中医内科杂志,2011,25(2):34-34.Wang JX,Yang GL.Features of stroke and interpretation of Chest diseases[J].Journal of Practical Traditional Chinese Internal Medicine,2011,25(2):34-44.
[15] 张红珍.浅谈动脉粥样硬化与虚瘀痰浊[J].新中医,2012,44(8):173-175.Zhang HZ.Discussion on atherosclerosis and deficien cy and stagnation of phlegm[J].Journal of New Chi nese Medicine,2012,44(8):173-175.
[16] Holmes K,Roberts OL,Thomas AM,et al.Vascular en dothelial growth factor receptor-2:structure,function,intracellular signaling and therapeutic inhibition[J].Cel lular Signaling,2007,19(10):2003-2012.
[17] 刘利妍,刘向群,陈效安,等.大鼠动脉粥样硬化形成过程中TAFI水平变化及作用机制[J].山东大学学报(医学版),2007,45(8):787-791.Liu LY,liu XQ,Chen XA,et al.TAFI level and its mech anism in the process of atherosclerosis in rats[J].Jour nal of Shandong University (Health Sciences),2007,45(8):787-791.
[18] Guo M,Sahni SK,Sahni A,et al.Fibrinogen regulates the expression of inflammatory chemokines through NF-kappaB activation of endothelial cells[J].Thrombo sis & Haemostasis,2004,92(4):858-866.
[19] 左丽,周鹏.血浆LOX-1与人外周血单核细胞LOX-1 mRNA表达水平与冠脉病变程度的相关性研究[J].川北医学院学报,2013,33(6):807-811.Zuo L,Zhou P.The relationship between angiographic coronary artery lesions and the expression of serum lox-1 and lox-1 mRNA on circulating monocytes[J].Journal of North Sichuan Medical College,2013,33(6):807-811.
[20] 王伟,黄静妍,谭金晶,等.Toll样受体5基因遗传多态性与中风风痰瘀阻证显著关联的探讨[J].辽宁中医杂志,2016,43(12):2495-2497.Wang W,Huang JY,Tan JJ,et al.Genetic polymor phism of toll-like receptor 5 gene significantly associat ed with stroke wind-phlegm-stasis syndrome[J].Liaon ing Journal of Traditional Chinese Medicine,2016,43(12):2495-2497.
[21] 陈琪.β纤维蛋白原-455G/A基因多态性与冠心病的关系[J].中国动脉硬化杂志,2013,21(10):931-934.Chen Q.Association of β-455G/A polymorphism with coronary heart disease[J].Chinese Journal of Atherosclerosis,2013,21(10):931-934.
[22] 何娴,叶晓冰,陈家钖,等.脑卒中患者载脂蛋白E基因多态性与血小板水平相关性研究[J].黑龙江医药,2018,31(1):118-120.He X,Ye XB,Chen JD,et al.Correlation between apoli poprotein E gene polymorphism and platelet level in stroke patients [J].Heilongjiang pharmaceutical,2018,31(1):118-120.
[23] 周明权.冠心病患者凝血四项及纤维蛋白原多态性分析[J].中国实用医药,2012,7(5):70-71.Zhou MQ.Analysis of four clotting items and fibrinogen polymorphism in patients with coronary heart disease[J].Chinese Journal of Practical Medicine,2012,7(5):70-71.
[24] 袁蓉,王阶,郭丽丽.冠心病痰瘀互结证的近代研究及中医治疗进展[J].中国中药杂志,2016,41(1):35-37.Yuan R,Wang T,Guo LL.Advances in modern research and TCM treatment of phlegm and blood stasis syn drome of coronary heart disease[J].Chinese Journal of Traditional Chinese Medicine,2016,41 (1):35-37.
[25] 任建勋,刘建勋,林成仁,等.冠心病不稳定型心绞痛痰瘀互结证临床生化指标研究[J].中医杂志,2012,53(8):665-668.Ren JX,Liu JX,Lin CR,et al.The biochemical indicators in coronary heart disease patients with unstable angina pectoris[J].Journal of Traditional Chinese Medicine,2012,53(8):665-668.