IL-6基因启动子区-572G/C、-597G/A多态性的实时荧光PCR检测及其与汉族人冠心病的关系
|
摘要
目的
为了研究IL-6基因多态性与心血管病的相关性，根据荧光共振能量转移和溶点曲线分析原理，建立了一种IL-6基因快速分型的实时荧光分析方法。并通过对中国北方地区汉族人IL-6基因启动子区-572G／C，-597G／A多态性与体重指数(BMI)和炎症因子等生化指标的相关性的调查，探索基因多态性与冠心病(CHD)发生发展的关系。
方法
利用实时荧光PCR方法检测我国北方地区汉族人群IL-6基因启动子-572G／C，-597G／A的多态性。甘油三脂(TG)、总胆固醇(TC)采用酶法测定。高密度脂蛋白胆固醇(HDL)、低密度脂蛋白胆固醇(LDL)采用均相酶法测定。脂蛋白a[Lp(a)]、载脂蛋白A1(ApoA1)、载脂蛋白A2(ApoA2)、载脂蛋白B(ApoB)、载脂蛋白C2(ApoC2)、载脂蛋白C3(ApoC3)、载脂蛋白E(ApoE)采用免疫透射比浊法测定。超敏C反应蛋白(hsCRP)采用颗粒增强的免疫散射比浊法测定。白细胞介素-6(IL-6)采用细胞因子芯片检测。通过实时荧光PCR方法、直接DNA测序、聚合酶链反应-限制性片段长度多态性(RFLP)三种检测方法的比较，分析其对77例样本检测结果的一致性。
结果
一、在国内首次成功建立了同时检测IL-6基因启动子区-572G／C、-597G／A多态性的实时荧光PCR检测方法。用DNA测序方法验证检测结果正确。经对中国北方地区汉族123例健康对照和194例冠心病患者的IL-6基因检测，发现在-597位点仅有两种基因型，为GG型和GA型，尚未发现AA型。-572位点有三种基因型，分别为GG，GC，CC型。其中-597GA型为国内首次报道。
二、实时荧光PCR方法、直接DNA测序、RFLP三种检测方法的比较分析发现：在77例样本中，采用实时荧光PCR方法与直接DNA测序相比仅有3例结果不一致。77例PCR产物用限制性内切酶BsrBI酶切，有20例与实时荧光PCR方法结果不一致。当PCR产物用限制性内切酶FokI酶切时，仅有5例是GA型，另外两例未被检测出。
三、统计分析发现IL-6基因启动子区-572G／C基因型频率和等位基因频率在冠心病组和对照组中的分布差异无统计学意义。携带G等位基因(GG+GC)和非G等位基因(CC)频率在冠心病组和对照组中的差异有统计学意义。在正常对照组中，与非G等位基因相比，携带G等位基因组的收缩压中位数水平显著升高。在所有观测对象中，与非G等位基因相比，携带G等位基因组的BMI、hsCRP、收缩压中位数水平显著升高。
四、Logistic回归分析显示，ApoC2、TH、lip(a)、年龄、TG、性别、高血压是本研究对象冠心病发生的独立危险因素，ApoAl是保护因子，未见G等位基因是一个独立的危险因子。
结论
实时荧光PCR方法在30分钟内能够完成32例样本两个位点的同时检测。与DNA测序、RFLP方法相比，具有简单、准确、快速的特点，便于临床实验室大规模样本筛查。本研究未发现IL-6基因启动子区-597G／A多态性与CHD的易感性有关，但发现-572G／C多态性与CHD的易感性有关。其机制可能与-572G／C多态性可导致BMI、hsCRP和血压的变化有关。
Objective
In order to explore the associations between IL-6 gene polymorphisms and CHD, a rapid genotyping assay for the IL-6 gene by fluorescent resonance energy transfer (FRET) and melting curves was developed. The association between polymorphisms and CHD was explored by investigating the polymorphisms (-572G/C, -597G/A) in IL-6 gene promoter area and body mass index (BMI), inflammatory factors and other biochemistry parameters in Han nationality of North China.
Methods
We described a real-time polymerase chain reaction method for detecting the -572G/C and -597G/A promoter polymorphisms of IL-6 gene in Han nationality of North China. Serum TC and TG levels were determined with standard enzymic colorimetric methods. Serum HDL and LDL levels were determined with homogeneous enzymatic colorimetric assay. Concetrations of Lp (a), ApoA1, ApoA2, ApoB, ApoC2, ApoE and ApoC3 were measured by immuno-turbidirnetric assay (ITA). Serum hsCRP were determined by particle enhanced immunonephelometry. Serum IL-6 levels were detected by Cytokine panel. The concordance of 77 samples were analysed by real-time PCR, DNA sequence analysis and restriction fragment length polymorphism (RFLP) assay.
Results
A real-time PCR method was developed successfully and DNA sequencing analysis validated the results. 190 CHD patients and 123 controls were analysed. The data are the first time to show that genetype of IL-6 gene promoter -597G/A polymorphism in 7 cases were GA and were GG in others, whereas no AA
genotype had been found. GG, GC, and CC genotypes were also found of IL-6 gene -572G/C polymorphisms.
Compated real-time PCR method with DNA sequence analysis, we found that only three samples were different in 77 samples. However, when the PCR product of 77 samples was digested by restriction enzyme BsrBI, 20 of them were different from real-time PCR method. The PCR product was digested by restriction enzyme Fok I, only five cases were found to be the GA genotype.
Statistc analysis shows that no difference had been found between the frequencies of IL-6 gene -572G/C genotypes and alleles in CHD and control group. Significant difference was found between the G allele carrier (GG+GC) and non-G allele carrier (CC) in CHD group and control group. In the control group, median levels of systolic blood pressure of G allele carriers were significantly higher than that of non-G allele carriers. Among all the subjects, BMI, hsCRP and systolic blood pressure in the group of G allele carrier were significant higher than the group of non-G allele carrier.
Multivariate logistic regression analysis showed that ApoC2, TH, lip (a), age, TG, sex and high blood pressure were risk factors for CHD. ApoAl was a protective factor. G allele was not found to be an independent risk factor.
Conclusion
Two mutation sites of 32 samples could be detected simultaneously within 30 min. Compared with RFLP analysis and DNA sequencing analysis, it is a simple, accurate, and fast method, which is more suitable for large-scaled screen in clinical laboratory. The results indicated that at the two mutation sites, only the IL-6 gene -572G/C polymorphism might be correlated with susceptibility to CHD in Han nationality of North China. The mechanism may be related with the changes of BMI, hsCKP and blood pressure levels resulted from the polymorphism IL-6 -572G/C.
为了研究IL-6基因多态性与心血管病的相关性，根据荧光共振能量转移和溶点曲线分析原理，建立了一种IL-6基因快速分型的实时荧光分析方法。并通过对中国北方地区汉族人IL-6基因启动子区-572G／C，-597G／A多态性与体重指数(BMI)和炎症因子等生化指标的相关性的调查，探索基因多态性与冠心病(CHD)发生发展的关系。
方法
利用实时荧光PCR方法检测我国北方地区汉族人群IL-6基因启动子-572G／C，-597G／A的多态性。甘油三脂(TG)、总胆固醇(TC)采用酶法测定。高密度脂蛋白胆固醇(HDL)、低密度脂蛋白胆固醇(LDL)采用均相酶法测定。脂蛋白a[Lp(a)]、载脂蛋白A1(ApoA1)、载脂蛋白A2(ApoA2)、载脂蛋白B(ApoB)、载脂蛋白C2(ApoC2)、载脂蛋白C3(ApoC3)、载脂蛋白E(ApoE)采用免疫透射比浊法测定。超敏C反应蛋白(hsCRP)采用颗粒增强的免疫散射比浊法测定。白细胞介素-6(IL-6)采用细胞因子芯片检测。通过实时荧光PCR方法、直接DNA测序、聚合酶链反应-限制性片段长度多态性(RFLP)三种检测方法的比较，分析其对77例样本检测结果的一致性。
结果
一、在国内首次成功建立了同时检测IL-6基因启动子区-572G／C、-597G／A多态性的实时荧光PCR检测方法。用DNA测序方法验证检测结果正确。经对中国北方地区汉族123例健康对照和194例冠心病患者的IL-6基因检测，发现在-597位点仅有两种基因型，为GG型和GA型，尚未发现AA型。-572位点有三种基因型，分别为GG，GC，CC型。其中-597GA型为国内首次报道。
二、实时荧光PCR方法、直接DNA测序、RFLP三种检测方法的比较分析发现：在77例样本中，采用实时荧光PCR方法与直接DNA测序相比仅有3例结果不一致。77例PCR产物用限制性内切酶BsrBI酶切，有20例与实时荧光PCR方法结果不一致。当PCR产物用限制性内切酶FokI酶切时，仅有5例是GA型，另外两例未被检测出。
三、统计分析发现IL-6基因启动子区-572G／C基因型频率和等位基因频率在冠心病组和对照组中的分布差异无统计学意义。携带G等位基因(GG+GC)和非G等位基因(CC)频率在冠心病组和对照组中的差异有统计学意义。在正常对照组中，与非G等位基因相比，携带G等位基因组的收缩压中位数水平显著升高。在所有观测对象中，与非G等位基因相比，携带G等位基因组的BMI、hsCRP、收缩压中位数水平显著升高。
四、Logistic回归分析显示，ApoC2、TH、lip(a)、年龄、TG、性别、高血压是本研究对象冠心病发生的独立危险因素，ApoAl是保护因子，未见G等位基因是一个独立的危险因子。
结论
实时荧光PCR方法在30分钟内能够完成32例样本两个位点的同时检测。与DNA测序、RFLP方法相比，具有简单、准确、快速的特点，便于临床实验室大规模样本筛查。本研究未发现IL-6基因启动子区-597G／A多态性与CHD的易感性有关，但发现-572G／C多态性与CHD的易感性有关。其机制可能与-572G／C多态性可导致BMI、hsCRP和血压的变化有关。
Objective
In order to explore the associations between IL-6 gene polymorphisms and CHD, a rapid genotyping assay for the IL-6 gene by fluorescent resonance energy transfer (FRET) and melting curves was developed. The association between polymorphisms and CHD was explored by investigating the polymorphisms (-572G/C, -597G/A) in IL-6 gene promoter area and body mass index (BMI), inflammatory factors and other biochemistry parameters in Han nationality of North China.
Methods
We described a real-time polymerase chain reaction method for detecting the -572G/C and -597G/A promoter polymorphisms of IL-6 gene in Han nationality of North China. Serum TC and TG levels were determined with standard enzymic colorimetric methods. Serum HDL and LDL levels were determined with homogeneous enzymatic colorimetric assay. Concetrations of Lp (a), ApoA1, ApoA2, ApoB, ApoC2, ApoE and ApoC3 were measured by immuno-turbidirnetric assay (ITA). Serum hsCRP were determined by particle enhanced immunonephelometry. Serum IL-6 levels were detected by Cytokine panel. The concordance of 77 samples were analysed by real-time PCR, DNA sequence analysis and restriction fragment length polymorphism (RFLP) assay.
Results
A real-time PCR method was developed successfully and DNA sequencing analysis validated the results. 190 CHD patients and 123 controls were analysed. The data are the first time to show that genetype of IL-6 gene promoter -597G/A polymorphism in 7 cases were GA and were GG in others, whereas no AA
genotype had been found. GG, GC, and CC genotypes were also found of IL-6 gene -572G/C polymorphisms.
Compated real-time PCR method with DNA sequence analysis, we found that only three samples were different in 77 samples. However, when the PCR product of 77 samples was digested by restriction enzyme BsrBI, 20 of them were different from real-time PCR method. The PCR product was digested by restriction enzyme Fok I, only five cases were found to be the GA genotype.
Statistc analysis shows that no difference had been found between the frequencies of IL-6 gene -572G/C genotypes and alleles in CHD and control group. Significant difference was found between the G allele carrier (GG+GC) and non-G allele carrier (CC) in CHD group and control group. In the control group, median levels of systolic blood pressure of G allele carriers were significantly higher than that of non-G allele carriers. Among all the subjects, BMI, hsCRP and systolic blood pressure in the group of G allele carrier were significant higher than the group of non-G allele carrier.
Multivariate logistic regression analysis showed that ApoC2, TH, lip (a), age, TG, sex and high blood pressure were risk factors for CHD. ApoAl was a protective factor. G allele was not found to be an independent risk factor.
Conclusion
Two mutation sites of 32 samples could be detected simultaneously within 30 min. Compared with RFLP analysis and DNA sequencing analysis, it is a simple, accurate, and fast method, which is more suitable for large-scaled screen in clinical laboratory. The results indicated that at the two mutation sites, only the IL-6 gene -572G/C polymorphism might be correlated with susceptibility to CHD in Han nationality of North China. The mechanism may be related with the changes of BMI, hsCKP and blood pressure levels resulted from the polymorphism IL-6 -572G/C.
引文
1 Ross R. Atherosclerosis is an inflammatory disease. Am Hart J. 1999 Nov; 138(5 Pt 2):S419-20.
2 Blake GJ, Ridker PM. Inflammatory bio-markers and cardiovascular risk prediction. J Intern Med 2002; 252:283-294.
3 Libby P, Ridker PM. Inflammation and atherosclerosis: role of C-reactive protein in risk assessment. Am J Med 2004; 111:9S-16S.
4 Mestries C, Kruithof EKO, Gascon MP, Herodin F, Agay D, Ythier A. In vivo modulation of coagulation and fibrinolysis by recombinant glycosylated human interleukin-6 in baboons. Eur Cytokine Netw. 1994; 5(3): 275-81.
5 Bataille R, Klein B. C-reactive protein levels as a direct indicator of interleukin-6 levels in humans in vivo. Arthritis Rheum. 1992; 35: 982-983.
6 Blake GJ, Ridker PM. C-reactive protein and other inflammatory risk markers in acute coronary syndromes. J Am Coll Cardiol 2003; 41:37S-42S.
7 Willerson JT, Ridker PM. Inflammation as a cardiovascular risk factor. Circulation 2004; 109:2-10.
8 Pearson TA, Mensah GA, Alexander RW, et al. Markers of Inflammation and Cardiovascular Disease: apolication to clinicaland publilc health practice: A statement for healthcare profseeional from the Centrs for Disease Control and Prevention and the American Heart Association. Ciculation.2003; 107:499-511.
9 Fishman D, Faulds G, Jeffery R, et al .The effect of novel in polymorphism in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 level and an association with systemic-onset juvenile chronic arthritis. J Clin Invest (1998) 102: 1369-1376.
10 Biasucci LM, Liuzzo G, Fantuzzi G, Caligiuri G, Rebuzzi AG, Ginnetti F, et al. Increasing concentrations of interleukin (IL)-lRa and IL-6 during the first 2 days of hospitalization in unstable angina are associated with increased risk of in-hospital coronary events. Circulation 1999; 99:2079-2084.
11 RidkerPM, Rifai N, Stampfer MJ, Hennekens CH. Plasma concentration of interleukin-6 and the risk of future myocardial infarction among apparently healthy men. Circulation 2000; 101:1767-1772.
12 Cesari M, Penninx BW, Newman AB, Kritchevsky SB, Nicklas BJ, Sutton-Tyrrell K, et al. Inflammatory markers and onset of cardiovascular events: results from the Health ABC study. Circulation 2003; 108:2317-2322.
13 Catherine F. Terry, Valerie Loukaci, Fiona R.Green. Cooperative influence of genetic polymorphisms on interleukin-6 transcriptional regulation J. Biol. Chem., Vol. 275, Issue 24, 18138-18144, June 16, 2000.
14 Humphies SE, Luong LA, Ogg MS, et al. The interleukin-6-174G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in health men [J], Eur Heart J, 2001, 22; 2243-2252.
15 Jones KG, Brull DJ, Brown LC, Sian M, Greenhalgh RM, Humphies SE, Powell JT. Interleukin-6 (IL-6) and the prognosis of abdominal aortic aneurysms. Circulation 2001; 103:2260-5.
16 Brull DJ, Montgomery HE, Sanders J, et al. Interleukin-6 gene -174G＞C and-572G＞C promoter polymorphism are strong predictors of plasma interleukine-6 leveles after coronary artery bypass surgery. Arterioscler. Thromb Vasc. Biol. 2001, vol. 21, 1458-1463.
17 Humphrise SE, Luong LA, Ogg MS, et al. The interleukin-6-174G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in health men [J], Eur Heart J, 2001, 22; 2243-2252.
18 Chun Soo Lin, Shouhuan Zheng, Yon Su Kim, et al. Suhnggwon Kim, Jung Sang Lee, Dong-Wan Chae. The-174 G to C polymorphism of interleukin-6 gene is very rare in Koreans. Cytokine. 2002, 19 (1) 52-54.
19 付海霞，张嘉莹，李庚山等．白细胞介素6基因-572C／G多态性与心肌梗塞易感性的关联研究．中华医学遗传学杂志．2006，23(3)245-249．
20 Hojo Y, Ikeda U, Katsuki T, et al. Interleukin6 expression in coronary circulation after coronary angioplasty as a risk factor for restenosis. heart, 2000,84:83-87.
21 Marz W, Wieland H. HMG-reductase inhibition: anti-inflammatory effects beyond lipid lowering. Herz, 2000, 25:117-125.
22 Terry CF, Loukaci V, Green FR. Cooperative influence of genetic polymorphisms on intedeukin-6 transcriptional regulation. Jour of Bio Chem. 2000, 275 (24); 18-138.
23 Bertsch T, ZimmerW, CasarinW. Real-Time PCR Assay with Fluorescent Hybridization Probes for Rapid Interleukin-6 Promoter (-174G＞C) Genotyping. ClinChem, 2001, 47(10): 1873-4.
24 贾玉艳，陈宏．SNP分子标记的研究及应用[J]．黄牛杂志，2003，29(1)：42-45．
25 罗怀容，施鹏，张亚平．单核苷酸多态性的研究技术[J]．遗传，2001，23(5)：471-476．
26 Marz W, Wieland H. HMG-CoA reductase inhibition: antiinflammatory effects beyond lipid lowering. Herz, 2000, 25:117-125.
27 中华医学会心血管病分会，中华心血管杂志编辑委员会，中国循环杂志编辑委员会．急性心肌梗死诊断和治疗指南．中华心血管病杂志，2001．29：710-725．
28 Marile Bennermo, Claes Held, et al. Prognostic value of plasma interleukin-6 concentrations and the -174G＞C and -572G＞C promoter polymorphisms of the interleukin-6 gene in patients with acute myocardial infarction treated with thrombolysis. J Atherosclerosisl.2004, 74, 157-163.
29 Chung HW, Seo JS, Hur SE, et al. Association of interleukin-6 promoter variant with bone mineral density in pre-menopausal women. J Hum Genet, 2003, 48(5): 243-8.
30 付海霞，李庚山，李艳等．白细胞介素6基因多态性对冠心病发病易感性的影响及其机制．中华心血管病杂志．2006，34(6)519-522．
31 付海霞，李庚山，李艳等．IL-6基因-597G／A与-572G／C多态性在湖北 汉族人群中的分布．武汉大学学报2004，25(2)194-197．
32 Wu,-D-M;Chu,-N-F;Shen,-M-H, et al.Obesity, plasma high sensitivity C- reactive protein levels and insulin resistance status among school children in Taiwan., Clin-Biochem. 2006 Aug; 39(8): 810-5.
33 Cummings, -D-M; King, -D-E; Mainous, -A-G; etal. Combining serum biomarkers: the association of C-reactive protein, insulin sensitivity, and homocysteine with cardiovascular disease history in the general US population., Eur-J-Cardiovasc-Prev-Rehabil. 2006 Apr; 13(2): 180-5.
34 Clement, -K; Viguerie, -N; Poitou, -C; etal. Weightloss regulates inflammation -related genes in white adipose tissue of obese subjects. FASEB-J. 2004 .Nov; 18(14): 1657-69.
35 Humphies SE, Luong LA, Ogg MS, et al. The interleukin-6 -174G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in health men [J], Eur Heart J, 2001, 22; 2243-2252.
1 Joes EY, Stuart DI, Walker NP. Structure of tumour necrosis factor. Nature. 1989, 338(6212): 225-228
2 Bazan JF, Timans JC, Kastelein RA. A newly defined interleukin-1? Nature. 1996, 379(6566): 591.
3 Thornberry NA, Bull HG, Calaycay JR, et al. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature. 1992, 356(6372): 768-774.
4 Miller MD, Krangel MS. Biology and biochemistry of the chemokines: a family of chemotactic and inflammatory cytokines. Crit Rev Immunol. 1992, 12(1-2): 17-46.
5 Bischoff SC, Krieger M, Brunner T, et al. Monocyte chemotactic protein 1 is a potent activator of human basophils. J Expt Med. 1992, 175(5): 1271-1275.
6 Hack CE, Aarden LA, Thijs LG.. Role of cytokines in sepsis. Adv Immunol. 1997, 66: 101-195.
7 Saadeddin SM, Habbab MA, Ferns GA, et al. Med Sci Monit, 2002,8(1): RA 5-12.
8 Koukkunen H, Penttila K, Kemppainen A, et al. C-reactive protein fibrinogen, interleukin-6 and tumor necrosis factor alpha in the prognostic classification of unstable angina pectoris. Ann Med, 2001, 33:37-47.
9 Lagrand WK, Niessen HW, Wolbink GJ, et al. C-reactive protein colocaliaes with complement in human heart during acute myocardial infarction. Circulation. 1997 Jan 7, 95(1): 97-103.
10 BarathP, Fishbein MC, CaoJ, et al. Tumor necrosis factor gene expression in human vascular intimal smooth muscle cells detected by insitu hybridization. Am J Pathol, 1990 Sep; 137(3):503-509.
11 BarathP, Fishbein MC, CaoJ, et al. Detection and localization of Tumor necrosis factor in human atheroma. Am J Cardiol, 1990 Feb 1; 65(5): 297-302.
12 A.G Wilson, J.A. Symons, T.L. McDowell, et al. Effects of a polymorphism in the tumor necrosis factor a promoter on transcriptional activation. Proc. Natl. Acad. Sci. USA 94 (1997), pp. 3195-3199.
13 Vendrell J,Femandez-Real JM, Gutierrez C, etal. A polymorphism in the promoter of the tumor necrosis factor-alpha gene (-308) is associated with coronary heart disease in type2 diabetic patients. Atherosclerosis. 2003 Apr, 167(2): 257-64.
14 Dendoussis GV, Panagiotakos DB, Vidra NV, etal. Association between TNF-alpha -308G>A polymorphism and the development of acute coronary syndromes in Greek subjects: The CARDIO2000-GENE Study. Genet Med. 2005 July-August; 7(6): 411-416.
15 Li Y, Wang M, Zhang PA, et al, Association between tumor necrosis factor-beta polymorphisms and coronary heart disease in a Chinese population. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2004 Dec, 21(6): 583-6.
16 Hojo Y, Ikeda U, Katsuki T, etal.interleukin6 expression in coronary circulation after coronary angioplasty as a risk factor for restenosis. Heart, 2000, 84:83-87.
17 Seino Y, I keda M, Ymamato K, et al.interleukin-6 gene transerpts are expressed in human atherosclerotic lesion [J]. Cytokine, 1994, 26:141.
18 Marz W, Wieland H.HMG-reductase inhibition: anti-inflammatory effects beyond lipid lowering. Herz, 2000, 25:117-125.
19 Terry CF, Loukaci V, Green FR. Cooperative influence of genetic polymorphisms on interleukin-6 transcriptional regulation. Jour of Bio Chem, 2000,275 (24); 18-138.
20 Markus N, Bernhard R, Winkelmann MM, et al. The interleukin-6 -174G/C promoter polymorphism in the LURIC cohort: no association with plasma interleukin-6, coronary artery disease, and myocardial infarction [J]. J Mol Med, 2002, 80; 507-513.
21 Humphrise SE, Luong LA, Ogg MS, et al. The interleukin-6 -174G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in health men [J], Eur Heart J, 2001, 22; 2243-2252.
22 Fishman D, Faulds G, Jeffery R, et al. The effect of novel in polymorphism in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 level and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 1998, 102: 1369-1376.
23 Mendall MA, Patel P, Asante M, et al.1997 Relation of serum cytokine concentration to cardiovascular risk factors and coronary artery disease. Heart. 11:273-277.
24 Baggio G, Donazzan S, Monti D, et al.1998 Liorprotein (a) and lipoprotein profile in healthy centenarians; a reappraisal of vascular risk factors. FASEB12: 433-437.
25 Jose-Manuel, Fernandez-Real, Montserrat Broch et al .2000 Interleukin-6 Gene Polymorphism and Lipid Abnormalities in healthy subjects.
26 Austin MA, Hokanson JE, Edwards KL. Hypertriglyceridemia as a cardiovascular risk factor.Am J Cardiol 1998;81(4A);7B-12B.
27 Haim M, Benderly M, Brunner D, et al. Elevated serum triglyceride levels and long-term mortality in patients with coronary heart disease: the Bezafibrate Infarction Prevention (BIP) Registry. Circulation, 1999; 100(5): 475-482.
28 Brull DJ, Montogomery HE, Sanders J, et al. Interleukin-6 gene -174G>C and -572G>C promoter polymorphism are strong predictors of plasma interleukin-6 levels after coronary artery bypass surgery. Arterioscler Thromb Vasc Biol 2001, 21:1458-63.
29 Marile Bennermo, Claes Held, et al. Prognostic value of plasma interleukin-6 concentrations and the -174G＞C and -572G＞C promoter polymorphisms of the interleukin-6 gene in patients with acute myocardial infarction treated with thrombolysis. J Atherosclerosis 2004, 174:157-163.
30 Chung HW, Seo JS, Hur SE, et al. Association of intefleukin-6 promoter variant with bone mineral density in pre-menopausal women. J Hum Genet,2003, 48(5): 243-8.
31 Chun Soo Lin, Shouhuan Zheng, Yon Su Kim, et al. Suhnggwon Kim, Jung Sang Lee, Dong-Wan Chae. The-174 G to C polymorphism of interleukin-6 gene is very rare in Koreans. Cytokine. 2002, 19 (1) 52-54.
32 韦叶生，汪明，覃志坚等．白细胞介素-6基因启动子-174G／C单核苷酸多态性的研究[J]右江民族医学院学报，2004，26(6)：782-785．
33 付海霞，李庚山，李艳等．IL-6基因-597G／A与-572C／G多态性在湖北汉族人群中的分布[J]武汉大学学报，2004，25(2)：194-196．
34 Biasucci L M, Liuzzo G, Fantuzzi G, et al. Increasing levels of interleukin (IL-1) Ra and IL-6 during the first 2 days of hospitalization in unstable angina are associated with increased risk of in -hospital coronary event. [J] Circulation, 1999 Apr, 99(16); 2079-2084.
35 Marczin N, Antonov A, Papapetropoulos A, et al. Monocyte induced aortic down regulation of nitric oxide synthase in cultured aortic endothelial cells. [J] Arterioscler Thromb Vasc Biol, 1996 Sep, 16 (9): 1095-1103.
36 BalbayY, Tikiz H, Baptiste RJ, etal. Circulating interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, and soluble ICAM-1 in patients with chronic stable angina and myocardial infarction. [J] Angiology. 2001 Feb, 52(2): 109-114.
37 Simon AD, Yazdani S,Wang W, etal. Circulating levels of IL-lbeta, a prothrombotic cytokine, are elevated in unstable angina versus stable angina. J Thromb Thrombolysis. 2000 Apr 9 (3): 217-222.
38 Wang YN, Che SM, Ma AQ, etal. Clinical significance of serum cytokines IL-1beta, sIL-2R, IL-6, TNF-alpha , and IFN-v in acute coronary syndrome. Chin Med Sci J. 2004 Jun, 19(2): 120-124.
39 Ikeda U. Inflammation and coronary artery disease. J Curr Vasc Pharmacol. 2003 Mar, 1(1): 65-70.
40 Aukrust P, Berge R K, Ueland T, etal. Interaction between chemokines and oxidative stress: possible pathogenic role in acute coronary syndromes. J Am Coll Cardiol, 2001 Feb, 37 (2): 485-491.
2 Blake GJ, Ridker PM. Inflammatory bio-markers and cardiovascular risk prediction. J Intern Med 2002; 252:283-294.
3 Libby P, Ridker PM. Inflammation and atherosclerosis: role of C-reactive protein in risk assessment. Am J Med 2004; 111:9S-16S.
4 Mestries C, Kruithof EKO, Gascon MP, Herodin F, Agay D, Ythier A. In vivo modulation of coagulation and fibrinolysis by recombinant glycosylated human interleukin-6 in baboons. Eur Cytokine Netw. 1994; 5(3): 275-81.
5 Bataille R, Klein B. C-reactive protein levels as a direct indicator of interleukin-6 levels in humans in vivo. Arthritis Rheum. 1992; 35: 982-983.
6 Blake GJ, Ridker PM. C-reactive protein and other inflammatory risk markers in acute coronary syndromes. J Am Coll Cardiol 2003; 41:37S-42S.
7 Willerson JT, Ridker PM. Inflammation as a cardiovascular risk factor. Circulation 2004; 109:2-10.
8 Pearson TA, Mensah GA, Alexander RW, et al. Markers of Inflammation and Cardiovascular Disease: apolication to clinicaland publilc health practice: A statement for healthcare profseeional from the Centrs for Disease Control and Prevention and the American Heart Association. Ciculation.2003; 107:499-511.
9 Fishman D, Faulds G, Jeffery R, et al .The effect of novel in polymorphism in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 level and an association with systemic-onset juvenile chronic arthritis. J Clin Invest (1998) 102: 1369-1376.
10 Biasucci LM, Liuzzo G, Fantuzzi G, Caligiuri G, Rebuzzi AG, Ginnetti F, et al. Increasing concentrations of interleukin (IL)-lRa and IL-6 during the first 2 days of hospitalization in unstable angina are associated with increased risk of in-hospital coronary events. Circulation 1999; 99:2079-2084.
11 RidkerPM, Rifai N, Stampfer MJ, Hennekens CH. Plasma concentration of interleukin-6 and the risk of future myocardial infarction among apparently healthy men. Circulation 2000; 101:1767-1772.
12 Cesari M, Penninx BW, Newman AB, Kritchevsky SB, Nicklas BJ, Sutton-Tyrrell K, et al. Inflammatory markers and onset of cardiovascular events: results from the Health ABC study. Circulation 2003; 108:2317-2322.
13 Catherine F. Terry, Valerie Loukaci, Fiona R.Green. Cooperative influence of genetic polymorphisms on interleukin-6 transcriptional regulation J. Biol. Chem., Vol. 275, Issue 24, 18138-18144, June 16, 2000.
14 Humphies SE, Luong LA, Ogg MS, et al. The interleukin-6-174G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in health men [J], Eur Heart J, 2001, 22; 2243-2252.
15 Jones KG, Brull DJ, Brown LC, Sian M, Greenhalgh RM, Humphies SE, Powell JT. Interleukin-6 (IL-6) and the prognosis of abdominal aortic aneurysms. Circulation 2001; 103:2260-5.
16 Brull DJ, Montgomery HE, Sanders J, et al. Interleukin-6 gene -174G＞C and-572G＞C promoter polymorphism are strong predictors of plasma interleukine-6 leveles after coronary artery bypass surgery. Arterioscler. Thromb Vasc. Biol. 2001, vol. 21, 1458-1463.
17 Humphrise SE, Luong LA, Ogg MS, et al. The interleukin-6-174G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in health men [J], Eur Heart J, 2001, 22; 2243-2252.
18 Chun Soo Lin, Shouhuan Zheng, Yon Su Kim, et al. Suhnggwon Kim, Jung Sang Lee, Dong-Wan Chae. The-174 G to C polymorphism of interleukin-6 gene is very rare in Koreans. Cytokine. 2002, 19 (1) 52-54.
19 付海霞，张嘉莹，李庚山等．白细胞介素6基因-572C／G多态性与心肌梗塞易感性的关联研究．中华医学遗传学杂志．2006，23(3)245-249．
20 Hojo Y, Ikeda U, Katsuki T, et al. Interleukin6 expression in coronary circulation after coronary angioplasty as a risk factor for restenosis. heart, 2000,84:83-87.
21 Marz W, Wieland H. HMG-reductase inhibition: anti-inflammatory effects beyond lipid lowering. Herz, 2000, 25:117-125.
22 Terry CF, Loukaci V, Green FR. Cooperative influence of genetic polymorphisms on intedeukin-6 transcriptional regulation. Jour of Bio Chem. 2000, 275 (24); 18-138.
23 Bertsch T, ZimmerW, CasarinW. Real-Time PCR Assay with Fluorescent Hybridization Probes for Rapid Interleukin-6 Promoter (-174G＞C) Genotyping. ClinChem, 2001, 47(10): 1873-4.
24 贾玉艳，陈宏．SNP分子标记的研究及应用[J]．黄牛杂志，2003，29(1)：42-45．
25 罗怀容，施鹏，张亚平．单核苷酸多态性的研究技术[J]．遗传，2001，23(5)：471-476．
26 Marz W, Wieland H. HMG-CoA reductase inhibition: antiinflammatory effects beyond lipid lowering. Herz, 2000, 25:117-125.
27 中华医学会心血管病分会，中华心血管杂志编辑委员会，中国循环杂志编辑委员会．急性心肌梗死诊断和治疗指南．中华心血管病杂志，2001．29：710-725．
28 Marile Bennermo, Claes Held, et al. Prognostic value of plasma interleukin-6 concentrations and the -174G＞C and -572G＞C promoter polymorphisms of the interleukin-6 gene in patients with acute myocardial infarction treated with thrombolysis. J Atherosclerosisl.2004, 74, 157-163.
29 Chung HW, Seo JS, Hur SE, et al. Association of interleukin-6 promoter variant with bone mineral density in pre-menopausal women. J Hum Genet, 2003, 48(5): 243-8.
30 付海霞，李庚山，李艳等．白细胞介素6基因多态性对冠心病发病易感性的影响及其机制．中华心血管病杂志．2006，34(6)519-522．
31 付海霞，李庚山，李艳等．IL-6基因-597G／A与-572G／C多态性在湖北 汉族人群中的分布．武汉大学学报2004，25(2)194-197．
32 Wu,-D-M;Chu,-N-F;Shen,-M-H, et al.Obesity, plasma high sensitivity C- reactive protein levels and insulin resistance status among school children in Taiwan., Clin-Biochem. 2006 Aug; 39(8): 810-5.
33 Cummings, -D-M; King, -D-E; Mainous, -A-G; etal. Combining serum biomarkers: the association of C-reactive protein, insulin sensitivity, and homocysteine with cardiovascular disease history in the general US population., Eur-J-Cardiovasc-Prev-Rehabil. 2006 Apr; 13(2): 180-5.
34 Clement, -K; Viguerie, -N; Poitou, -C; etal. Weightloss regulates inflammation -related genes in white adipose tissue of obese subjects. FASEB-J. 2004 .Nov; 18(14): 1657-69.
35 Humphies SE, Luong LA, Ogg MS, et al. The interleukin-6 -174G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in health men [J], Eur Heart J, 2001, 22; 2243-2252.
1 Joes EY, Stuart DI, Walker NP. Structure of tumour necrosis factor. Nature. 1989, 338(6212): 225-228
2 Bazan JF, Timans JC, Kastelein RA. A newly defined interleukin-1? Nature. 1996, 379(6566): 591.
3 Thornberry NA, Bull HG, Calaycay JR, et al. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature. 1992, 356(6372): 768-774.
4 Miller MD, Krangel MS. Biology and biochemistry of the chemokines: a family of chemotactic and inflammatory cytokines. Crit Rev Immunol. 1992, 12(1-2): 17-46.
5 Bischoff SC, Krieger M, Brunner T, et al. Monocyte chemotactic protein 1 is a potent activator of human basophils. J Expt Med. 1992, 175(5): 1271-1275.
6 Hack CE, Aarden LA, Thijs LG.. Role of cytokines in sepsis. Adv Immunol. 1997, 66: 101-195.
7 Saadeddin SM, Habbab MA, Ferns GA, et al. Med Sci Monit, 2002,8(1): RA 5-12.
8 Koukkunen H, Penttila K, Kemppainen A, et al. C-reactive protein fibrinogen, interleukin-6 and tumor necrosis factor alpha in the prognostic classification of unstable angina pectoris. Ann Med, 2001, 33:37-47.
9 Lagrand WK, Niessen HW, Wolbink GJ, et al. C-reactive protein colocaliaes with complement in human heart during acute myocardial infarction. Circulation. 1997 Jan 7, 95(1): 97-103.
10 BarathP, Fishbein MC, CaoJ, et al. Tumor necrosis factor gene expression in human vascular intimal smooth muscle cells detected by insitu hybridization. Am J Pathol, 1990 Sep; 137(3):503-509.
11 BarathP, Fishbein MC, CaoJ, et al. Detection and localization of Tumor necrosis factor in human atheroma. Am J Cardiol, 1990 Feb 1; 65(5): 297-302.
12 A.G Wilson, J.A. Symons, T.L. McDowell, et al. Effects of a polymorphism in the tumor necrosis factor a promoter on transcriptional activation. Proc. Natl. Acad. Sci. USA 94 (1997), pp. 3195-3199.
13 Vendrell J,Femandez-Real JM, Gutierrez C, etal. A polymorphism in the promoter of the tumor necrosis factor-alpha gene (-308) is associated with coronary heart disease in type2 diabetic patients. Atherosclerosis. 2003 Apr, 167(2): 257-64.
14 Dendoussis GV, Panagiotakos DB, Vidra NV, etal. Association between TNF-alpha -308G>A polymorphism and the development of acute coronary syndromes in Greek subjects: The CARDIO2000-GENE Study. Genet Med. 2005 July-August; 7(6): 411-416.
15 Li Y, Wang M, Zhang PA, et al, Association between tumor necrosis factor-beta polymorphisms and coronary heart disease in a Chinese population. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2004 Dec, 21(6): 583-6.
16 Hojo Y, Ikeda U, Katsuki T, etal.interleukin6 expression in coronary circulation after coronary angioplasty as a risk factor for restenosis. Heart, 2000, 84:83-87.
17 Seino Y, I keda M, Ymamato K, et al.interleukin-6 gene transerpts are expressed in human atherosclerotic lesion [J]. Cytokine, 1994, 26:141.
18 Marz W, Wieland H.HMG-reductase inhibition: anti-inflammatory effects beyond lipid lowering. Herz, 2000, 25:117-125.
19 Terry CF, Loukaci V, Green FR. Cooperative influence of genetic polymorphisms on interleukin-6 transcriptional regulation. Jour of Bio Chem, 2000,275 (24); 18-138.
20 Markus N, Bernhard R, Winkelmann MM, et al. The interleukin-6 -174G/C promoter polymorphism in the LURIC cohort: no association with plasma interleukin-6, coronary artery disease, and myocardial infarction [J]. J Mol Med, 2002, 80; 507-513.
21 Humphrise SE, Luong LA, Ogg MS, et al. The interleukin-6 -174G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in health men [J], Eur Heart J, 2001, 22; 2243-2252.
22 Fishman D, Faulds G, Jeffery R, et al. The effect of novel in polymorphism in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 level and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 1998, 102: 1369-1376.
23 Mendall MA, Patel P, Asante M, et al.1997 Relation of serum cytokine concentration to cardiovascular risk factors and coronary artery disease. Heart. 11:273-277.
24 Baggio G, Donazzan S, Monti D, et al.1998 Liorprotein (a) and lipoprotein profile in healthy centenarians; a reappraisal of vascular risk factors. FASEB12: 433-437.
25 Jose-Manuel, Fernandez-Real, Montserrat Broch et al .2000 Interleukin-6 Gene Polymorphism and Lipid Abnormalities in healthy subjects.
26 Austin MA, Hokanson JE, Edwards KL. Hypertriglyceridemia as a cardiovascular risk factor.Am J Cardiol 1998;81(4A);7B-12B.
27 Haim M, Benderly M, Brunner D, et al. Elevated serum triglyceride levels and long-term mortality in patients with coronary heart disease: the Bezafibrate Infarction Prevention (BIP) Registry. Circulation, 1999; 100(5): 475-482.
28 Brull DJ, Montogomery HE, Sanders J, et al. Interleukin-6 gene -174G>C and -572G>C promoter polymorphism are strong predictors of plasma interleukin-6 levels after coronary artery bypass surgery. Arterioscler Thromb Vasc Biol 2001, 21:1458-63.
29 Marile Bennermo, Claes Held, et al. Prognostic value of plasma interleukin-6 concentrations and the -174G＞C and -572G＞C promoter polymorphisms of the interleukin-6 gene in patients with acute myocardial infarction treated with thrombolysis. J Atherosclerosis 2004, 174:157-163.
30 Chung HW, Seo JS, Hur SE, et al. Association of intefleukin-6 promoter variant with bone mineral density in pre-menopausal women. J Hum Genet,2003, 48(5): 243-8.
31 Chun Soo Lin, Shouhuan Zheng, Yon Su Kim, et al. Suhnggwon Kim, Jung Sang Lee, Dong-Wan Chae. The-174 G to C polymorphism of interleukin-6 gene is very rare in Koreans. Cytokine. 2002, 19 (1) 52-54.
32 韦叶生，汪明，覃志坚等．白细胞介素-6基因启动子-174G／C单核苷酸多态性的研究[J]右江民族医学院学报，2004，26(6)：782-785．
33 付海霞，李庚山，李艳等．IL-6基因-597G／A与-572C／G多态性在湖北汉族人群中的分布[J]武汉大学学报，2004，25(2)：194-196．
34 Biasucci L M, Liuzzo G, Fantuzzi G, et al. Increasing levels of interleukin (IL-1) Ra and IL-6 during the first 2 days of hospitalization in unstable angina are associated with increased risk of in -hospital coronary event. [J] Circulation, 1999 Apr, 99(16); 2079-2084.
35 Marczin N, Antonov A, Papapetropoulos A, et al. Monocyte induced aortic down regulation of nitric oxide synthase in cultured aortic endothelial cells. [J] Arterioscler Thromb Vasc Biol, 1996 Sep, 16 (9): 1095-1103.
36 BalbayY, Tikiz H, Baptiste RJ, etal. Circulating interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, and soluble ICAM-1 in patients with chronic stable angina and myocardial infarction. [J] Angiology. 2001 Feb, 52(2): 109-114.
37 Simon AD, Yazdani S,Wang W, etal. Circulating levels of IL-lbeta, a prothrombotic cytokine, are elevated in unstable angina versus stable angina. J Thromb Thrombolysis. 2000 Apr 9 (3): 217-222.
38 Wang YN, Che SM, Ma AQ, etal. Clinical significance of serum cytokines IL-1beta, sIL-2R, IL-6, TNF-alpha , and IFN-v in acute coronary syndrome. Chin Med Sci J. 2004 Jun, 19(2): 120-124.
39 Ikeda U. Inflammation and coronary artery disease. J Curr Vasc Pharmacol. 2003 Mar, 1(1): 65-70.
40 Aukrust P, Berge R K, Ueland T, etal. Interaction between chemokines and oxidative stress: possible pathogenic role in acute coronary syndromes. J Am Coll Cardiol, 2001 Feb, 37 (2): 485-491.