吉林地区汉族人eNOS基因多态性与2型糖尿病合并高血压相关性的研究
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摘要
随着人民生活水平,劳动方式及饮食结构的改变,平均寿命的延长,糖尿病的发病率逐年增高。由于糖尿病对人体的危害仅次于癌症,故被称为现代疾病中的第二杀手。糖尿病患者一旦出现高血压病,并发症有加重的表现。因此,寻找和建立有效的糖尿病合并高血压发病风险预测和发病前诊断技术,在临床上具有重要实用价值。
本研究采用聚合酶链反应(PCR)和2%琼脂糖凝胶电泳法,检测吉林地区汉族人的一氧化氮合酶(eNOS)外显子7单核苷酸多态性(G894T)和内含子4可变数目串联重复序列(VNTR)插入/缺失多态性(4a/b)。进一步探讨一氧化氮合酶(eNOS)基因分布与糖尿病合并高血压的关系。
结果显示:(1)所选样本基因型频率经Hardy-Weinburg遗传平衡定律检验达到平衡,具有代表性。(2)eNOS基因第7外显子894G→T多态性与糖尿病合并高血压具有相关性。T等位基因是2型糖尿病合并高血压的危险因素。虽然DM组与NC组基因频率和等位基因频率有明显不同,但没有差异性。(3)eNOS基因第4内含子多态性与糖尿病具有相关性。a等位基因与2型糖尿病的发生有关。
Objective:To investigate the association between the single nucleitide polymorphism in exon 7(G894T) and the insert/deletion polymorphism of variable number of tandem repeat in intron 4(4a/b) of the endothelial nitric oxide synthase (eNOS) and type 2 diabetic patiens with hypertension in the Han population in jilin region.
Methods: This research is divided into research object totally 3 sets. The first group(A group) is the diabetic patiens without hypertension including 45 persons(17 male, 28 female, average age was 48士8.54); The second group(B group) is the diabetic patiens with hypertension including 35 persons(13 male, 22 female, average age was 51士7.46); The second group(NC group) is matched control including 36 persons(17male, 18 female, average age was 47士8.44);Carry on 3 sets to check contrast control study. The next step is abstracting template DNA, adopt the American Promega company produce of the DNA extract kit quickly abstracting person`s white cell DNA of peripheral blood. The G894T polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis.The 4a/b polymorphism was determined by polymerase chain reaction-variable number of tandem repeat (PCR-VNTR) analysis.the last step is 2% agarose gel electrophoresis was performed,the results were observed under ultraviolet light.
Result: (1)Hardy-Weinburg inherit quilibrium laws examination result is that each genotype frequency attains heredity balance and have a community representative . (2)The frequency of T allele and TG genetype at exon 7 of eNOS were significantly higher in the DM with hypertension group than that in matched control group;but there was not have no obvious difference between DM without hypertension and DM with hypertension.the result of DM without hypertension and matched control is the same. (3)The frequency of a allele at introns 4 of eNOS was significantly higher in DM with hypertension group and DM without hypertension than in matched control group. there was not have no obvious difference between DM without hypertension and DM with hypertension.
Conclusion: ( 1 ) Hardy-Weinburg inherit quilibrium laws examination result is that each genotype frequency attains heredity balance and have a community representative .(2)the 894G→T polymorphism at exon 7 of eNOS gene may be a predisposal factor for diabetic with hypertension in jilin.T allele is related to diabetic with hypertension in type-2 diabetes mellitus(3)a allele of the 4a/b polymorphism at introns 4 of eNOS gene is related to diabetic.
本研究采用聚合酶链反应(PCR)和2%琼脂糖凝胶电泳法,检测吉林地区汉族人的一氧化氮合酶(eNOS)外显子7单核苷酸多态性(G894T)和内含子4可变数目串联重复序列(VNTR)插入/缺失多态性(4a/b)。进一步探讨一氧化氮合酶(eNOS)基因分布与糖尿病合并高血压的关系。
结果显示:(1)所选样本基因型频率经Hardy-Weinburg遗传平衡定律检验达到平衡,具有代表性。(2)eNOS基因第7外显子894G→T多态性与糖尿病合并高血压具有相关性。T等位基因是2型糖尿病合并高血压的危险因素。虽然DM组与NC组基因频率和等位基因频率有明显不同,但没有差异性。(3)eNOS基因第4内含子多态性与糖尿病具有相关性。a等位基因与2型糖尿病的发生有关。
Objective:To investigate the association between the single nucleitide polymorphism in exon 7(G894T) and the insert/deletion polymorphism of variable number of tandem repeat in intron 4(4a/b) of the endothelial nitric oxide synthase (eNOS) and type 2 diabetic patiens with hypertension in the Han population in jilin region.
Methods: This research is divided into research object totally 3 sets. The first group(A group) is the diabetic patiens without hypertension including 45 persons(17 male, 28 female, average age was 48士8.54); The second group(B group) is the diabetic patiens with hypertension including 35 persons(13 male, 22 female, average age was 51士7.46); The second group(NC group) is matched control including 36 persons(17male, 18 female, average age was 47士8.44);Carry on 3 sets to check contrast control study. The next step is abstracting template DNA, adopt the American Promega company produce of the DNA extract kit quickly abstracting person`s white cell DNA of peripheral blood. The G894T polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis.The 4a/b polymorphism was determined by polymerase chain reaction-variable number of tandem repeat (PCR-VNTR) analysis.the last step is 2% agarose gel electrophoresis was performed,the results were observed under ultraviolet light.
Result: (1)Hardy-Weinburg inherit quilibrium laws examination result is that each genotype frequency attains heredity balance and have a community representative . (2)The frequency of T allele and TG genetype at exon 7 of eNOS were significantly higher in the DM with hypertension group than that in matched control group;but there was not have no obvious difference between DM without hypertension and DM with hypertension.the result of DM without hypertension and matched control is the same. (3)The frequency of a allele at introns 4 of eNOS was significantly higher in DM with hypertension group and DM without hypertension than in matched control group. there was not have no obvious difference between DM without hypertension and DM with hypertension.
Conclusion: ( 1 ) Hardy-Weinburg inherit quilibrium laws examination result is that each genotype frequency attains heredity balance and have a community representative .(2)the 894G→T polymorphism at exon 7 of eNOS gene may be a predisposal factor for diabetic with hypertension in jilin.T allele is related to diabetic with hypertension in type-2 diabetes mellitus(3)a allele of the 4a/b polymorphism at introns 4 of eNOS gene is related to diabetic.
引文
1.Sowers JR, Ep steinM. D iabetes mellitus and associated hypertension, vascular disease, and nephropathy: an update [J]. Hypertension, 1995, 26 (8) : 8692879.
2.陈国伟,郑宗锷.现代心脏内科学[M].长沙:湖南科学技术出版社,1997:1215.
3.张立权,丁绍平,胡东升.2型糖尿病合并高血压患者左心室结构和功能变化[J].岭南心血管病杂志,2007,13(6):214-216
4.周方明,祝之明,谭建聪等高血压合并糖尿病患者心脏结构与功能的变化特点。中国糖尿病杂志,2002,10(4):199-201.
5.白璐, 乔晶, 高晓虹等.2型糖尿病合并高血压对心脑血管疾病的影响[J]. 中国误诊学杂志,2007,6(3):1201-1202
6.程讯民,江时森,马瑞等高血压合并糖尿病对心肌微血管内皮细胞功能的影响[J] Shanghai Med[J].2007,30:162-164
7.Chobanian AV ,Bak ris GL ,Black HR, et al. Seventh Report of the joint National Committee on prevention,Detection, Evaluation, and Treatment of High Blood Pressure [ J ]. JAMA , 2003, 289 ( 12) :2560-2571
8.郭风劲,彭惠民,张政等糖尿病合并高血压患者血清NO、NOS 水平的研究[J] . 中国老年学杂志,2004,24(3):207-208
9.A ljada A ,Dandona P.Effect of insulin on human aorticendothelialnitric oxide synthase[J] Metabolism, 2000; 49 (2) : 147-150
10.沈稚舟主编.糖尿病慢性并发症.上海.上海医科大学出版社,1999,4-41
11.Schulz R , Triggle CR. Role of NO in vascular smooth muscle and cardiac muscle function [J ] . Trends Pharmacol Sci , 1994 ,15 (7) :255 - 259.。
12.PanzaJA, Casino PR,Badar DM,et al.Effect of increased availability of Endothelium-derived nitric oxide precursor on endothelinm depedent vascular relaxation in normal subject &in patients with essential htpertension.Circulation, 1993,87:1468-1474.
13.鄢定红,等 一氧化氮缺乏在高血压及心血管重构中的作用[J]高血压杂志, 2000, 8 (4) : 349-353
14.康建华,赵连友,张跃义高血压病患者血浆一氧化氮和内皮素的变化[J] .中国心血管研究杂志, 2004,2(1):26-27
15.Lushcher T F. Imbalance of endothelium derived relaxing and contracting factors : A new concept in hypertension[J ] . Am J Hypertens , 1990 , 3 (4) :317 - 330.
16.邓正华,邓剑,向成玉等.冠心病患者血浆NO和ET水平变化的临床研究[J]. 中国现代医学杂志, 2005,15(3):388-389
17.王峰,高超何,美安.冠心病患者血浆一氧化氮及一氧化氮合酶活力的研究[J]. 中华劳动卫生职业病杂志,2004,22(6) :419-421
18.李东霞,潘志红,张江蓉等.不同程度动脉硬化一氧化氮水平的变化及意义[J]. 实用心脑肺血管病杂志,2005,13(1):6-8
19.张金海,赵士超.心力衰竭与血浆一氧化氮及内皮素的相关性研究[J].医药论坛杂志,2004,25(23) :16-17
20.赵艳芳,邱红,刘萍等. 充血性心力衰竭患者的血浆一氧化氮改变及意义[J].急诊医学,1998,7(1) :37-38
21.Marletta MA. Nitric oxide synthsae structure and mechanism [J ]. J B iol Chem , 1993, 268: 12231-12234.
22.Moncada S, Higgs A , Fuchgott FR. International union of pharmacology nomenclature in nitric oxide research [ J ].Pharm acol R ev, 1997, 49: 1372142.
23.Marsden PA,Heng HHQ,Scherer SW, et al.Structure and chro-mosomal localization of the human constitutive endothelial nitric oxide synthase gene.J Biol Chem 1993;268 ( 23 )17478-17488。
24.Zanetti M , Katusic ZS , Obrien T. Expression and function of recombinant endothelial nitric oxide synthase in human endothelial cells. J Vasc Res ,2000 ,37 :4492456.
25.Wang XL,MahaneyMC,Sim AS, et al. Genetic contribution of the endothelial constitutive nitric oxide synthase gene to plasma nitric oxide levels. Arterioscler Thromb Vast Biol,1997, 17(11):3147-3153.
26. Lacolley P, Gautier S, Poirier 0, et al. Nitric oxide synthase gene polymorphisms,blood pressure and aortic stiffness in normotensive and hypertensive sub jects. J Hypertens, 1998, 16:31一35.
27.Miyamoto Y, Saito Y, Kajiyama, et al. Endothelial nitric oxide synthase gene is positively associated with essential hypertension.Hypertension, 1998, ,Tul;32 (1):3一8.
28.Yasujima M, Tsutaya S, Shoji M. Endothelial nitric oxide synthase gene polymorthism and hypertension. Rinabo Byoir,1998,46:1199一1204.
29.Jachymova M, HORKY k, BULTAS J, et al. Association of theGlu238Asp polymorphism in the endothelial nitric oxide synthase gene with essential hypertension. Resistant toconventional therapy. Biochem Biophys Res Commun, 2001, 284:426一430.
30.Shoji M, Tsutaya S, Saito R, et al. Positive association of endothelial nitric oxide synthase gene polymorphism with hypertension in northern Japan. Life Sci, 2000, 66:2557-2562.
31. Benjafield AV , Morris BJ . Association analyses of endothelial nitric oxide synthase gene polymorphism in essential hypertension. Am J Hypertens , 2000 , 13 : 994-998.
32.Kato N , Sugiyama T , Morita H ,et al. Lack of evidence forassociation between the endothelial nitric oxide synthase gene and hypertension. Hypertension ,1999 ,33 :933-936
33.Tsujita Y, Baba S , Yamauchi R ,et al. Association analyses between genetic polymorphisms of endothelial nitric oxide synthase gene and hypertension in Japanese : The Suita Study. J Hypertens , 2001 , 19 :1941 —1948.
34.李东宝,华 琦,皮 林.内皮型一氧化氮合酶基因G894T多态性与原发性高血压的相关关系[J]. 高血压杂志,2002, 12(4):326-330
35.刘海珍,哈黛文. 内皮型一氧化氮合酶基因894 G→T多态性与原发性高血压的关系研究[J].中国循环杂志,2002,17(10):361-363
36.Miyahara K, Kawamoto T , Sase K, et al. Cloning and structural characterization of the human endothelial nitric oxide synthase gene. Eur J Biochem , 1994 ,223 :7192726
37.Tsukada T , Yokoyam K, Arai T , et al. Evidence of association of the Enos gene polymorphism with plasma NO metabolite levels in humans. Biochem Biophys Res Commun, 1998 ,245 ,190～193
38.Uwabo J , Soma M , Nakayama T , et al. Association of a variable number of tandem repeats in the endothelial constitutive nitric oxide synthase gene with essential hypertension in Japane-. Es.Am J Hypertens , 1998 ,11 :1252128.
39.黄惠彬,林丽香,陈鸣钦. 内皮型一氧化氮合酶基因多态性与原发性高血压、2 型糖尿病的关系. 中华内分泌代谢杂志,2002 ,18 :16～19
2.陈国伟,郑宗锷.现代心脏内科学[M].长沙:湖南科学技术出版社,1997:1215.
3.张立权,丁绍平,胡东升.2型糖尿病合并高血压患者左心室结构和功能变化[J].岭南心血管病杂志,2007,13(6):214-216
4.周方明,祝之明,谭建聪等高血压合并糖尿病患者心脏结构与功能的变化特点。中国糖尿病杂志,2002,10(4):199-201.
5.白璐, 乔晶, 高晓虹等.2型糖尿病合并高血压对心脑血管疾病的影响[J]. 中国误诊学杂志,2007,6(3):1201-1202
6.程讯民,江时森,马瑞等高血压合并糖尿病对心肌微血管内皮细胞功能的影响[J] Shanghai Med[J].2007,30:162-164
7.Chobanian AV ,Bak ris GL ,Black HR, et al. Seventh Report of the joint National Committee on prevention,Detection, Evaluation, and Treatment of High Blood Pressure [ J ]. JAMA , 2003, 289 ( 12) :2560-2571
8.郭风劲,彭惠民,张政等糖尿病合并高血压患者血清NO、NOS 水平的研究[J] . 中国老年学杂志,2004,24(3):207-208
9.A ljada A ,Dandona P.Effect of insulin on human aorticendothelialnitric oxide synthase[J] Metabolism, 2000; 49 (2) : 147-150
10.沈稚舟主编.糖尿病慢性并发症.上海.上海医科大学出版社,1999,4-41
11.Schulz R , Triggle CR. Role of NO in vascular smooth muscle and cardiac muscle function [J ] . Trends Pharmacol Sci , 1994 ,15 (7) :255 - 259.。
12.PanzaJA, Casino PR,Badar DM,et al.Effect of increased availability of Endothelium-derived nitric oxide precursor on endothelinm depedent vascular relaxation in normal subject &in patients with essential htpertension.Circulation, 1993,87:1468-1474.
13.鄢定红,等 一氧化氮缺乏在高血压及心血管重构中的作用[J]高血压杂志, 2000, 8 (4) : 349-353
14.康建华,赵连友,张跃义高血压病患者血浆一氧化氮和内皮素的变化[J] .中国心血管研究杂志, 2004,2(1):26-27
15.Lushcher T F. Imbalance of endothelium derived relaxing and contracting factors : A new concept in hypertension[J ] . Am J Hypertens , 1990 , 3 (4) :317 - 330.
16.邓正华,邓剑,向成玉等.冠心病患者血浆NO和ET水平变化的临床研究[J]. 中国现代医学杂志, 2005,15(3):388-389
17.王峰,高超何,美安.冠心病患者血浆一氧化氮及一氧化氮合酶活力的研究[J]. 中华劳动卫生职业病杂志,2004,22(6) :419-421
18.李东霞,潘志红,张江蓉等.不同程度动脉硬化一氧化氮水平的变化及意义[J]. 实用心脑肺血管病杂志,2005,13(1):6-8
19.张金海,赵士超.心力衰竭与血浆一氧化氮及内皮素的相关性研究[J].医药论坛杂志,2004,25(23) :16-17
20.赵艳芳,邱红,刘萍等. 充血性心力衰竭患者的血浆一氧化氮改变及意义[J].急诊医学,1998,7(1) :37-38
21.Marletta MA. Nitric oxide synthsae structure and mechanism [J ]. J B iol Chem , 1993, 268: 12231-12234.
22.Moncada S, Higgs A , Fuchgott FR. International union of pharmacology nomenclature in nitric oxide research [ J ].Pharm acol R ev, 1997, 49: 1372142.
23.Marsden PA,Heng HHQ,Scherer SW, et al.Structure and chro-mosomal localization of the human constitutive endothelial nitric oxide synthase gene.J Biol Chem 1993;268 ( 23 )17478-17488。
24.Zanetti M , Katusic ZS , Obrien T. Expression and function of recombinant endothelial nitric oxide synthase in human endothelial cells. J Vasc Res ,2000 ,37 :4492456.
25.Wang XL,MahaneyMC,Sim AS, et al. Genetic contribution of the endothelial constitutive nitric oxide synthase gene to plasma nitric oxide levels. Arterioscler Thromb Vast Biol,1997, 17(11):3147-3153.
26. Lacolley P, Gautier S, Poirier 0, et al. Nitric oxide synthase gene polymorphisms,blood pressure and aortic stiffness in normotensive and hypertensive sub jects. J Hypertens, 1998, 16:31一35.
27.Miyamoto Y, Saito Y, Kajiyama, et al. Endothelial nitric oxide synthase gene is positively associated with essential hypertension.Hypertension, 1998, ,Tul;32 (1):3一8.
28.Yasujima M, Tsutaya S, Shoji M. Endothelial nitric oxide synthase gene polymorthism and hypertension. Rinabo Byoir,1998,46:1199一1204.
29.Jachymova M, HORKY k, BULTAS J, et al. Association of theGlu238Asp polymorphism in the endothelial nitric oxide synthase gene with essential hypertension. Resistant toconventional therapy. Biochem Biophys Res Commun, 2001, 284:426一430.
30.Shoji M, Tsutaya S, Saito R, et al. Positive association of endothelial nitric oxide synthase gene polymorphism with hypertension in northern Japan. Life Sci, 2000, 66:2557-2562.
31. Benjafield AV , Morris BJ . Association analyses of endothelial nitric oxide synthase gene polymorphism in essential hypertension. Am J Hypertens , 2000 , 13 : 994-998.
32.Kato N , Sugiyama T , Morita H ,et al. Lack of evidence forassociation between the endothelial nitric oxide synthase gene and hypertension. Hypertension ,1999 ,33 :933-936
33.Tsujita Y, Baba S , Yamauchi R ,et al. Association analyses between genetic polymorphisms of endothelial nitric oxide synthase gene and hypertension in Japanese : The Suita Study. J Hypertens , 2001 , 19 :1941 —1948.
34.李东宝,华 琦,皮 林.内皮型一氧化氮合酶基因G894T多态性与原发性高血压的相关关系[J]. 高血压杂志,2002, 12(4):326-330
35.刘海珍,哈黛文. 内皮型一氧化氮合酶基因894 G→T多态性与原发性高血压的关系研究[J].中国循环杂志,2002,17(10):361-363
36.Miyahara K, Kawamoto T , Sase K, et al. Cloning and structural characterization of the human endothelial nitric oxide synthase gene. Eur J Biochem , 1994 ,223 :7192726
37.Tsukada T , Yokoyam K, Arai T , et al. Evidence of association of the Enos gene polymorphism with plasma NO metabolite levels in humans. Biochem Biophys Res Commun, 1998 ,245 ,190～193
38.Uwabo J , Soma M , Nakayama T , et al. Association of a variable number of tandem repeats in the endothelial constitutive nitric oxide synthase gene with essential hypertension in Japane-. Es.Am J Hypertens , 1998 ,11 :1252128.
39.黄惠彬,林丽香,陈鸣钦. 内皮型一氧化氮合酶基因多态性与原发性高血压、2 型糖尿病的关系. 中华内分泌代谢杂志,2002 ,18 :16～19