【作者】 魏丹宏；

【导师】 单江；

【作者基本信息】 浙江大学 ， 内科学（心血管病）， 2001， 硕士

【摘要】 一、背景： 冠心病（CHD）是当今世界威胁人类健康的主要疾病之一，然而其发病机理迄今未明。多数学者认为凡涉及到动脉粥样硬化、血栓形成和血管收缩的因素均易致CHD的发生。eNOS是L-精氨酸、一氧化氮途径的关键酶。NO是所有硝基类扩血管药的活性成分，具有松弛血管平滑肌、舒张血管，并有抑制血小板聚集、粘附，抑制白细胞的趋化激活，抑制血管平滑肌细胞的增生、迁移及清除超氧自由基等重要作用。NO是心血管系统中抗冠状动脉粥样硬化、血栓形成，维持正常血管舒缩反应中必不可少的保护因子。生理状态下，血管内皮细胞能持续产生少量NO。NO产生量的减少或活性的下降均有可能参与CHD的发生与发展。因此，eNOS基因被列为CHD的一个侯选基因。有报道：人类血浆NO代谢产物水平与eNOS基因多态性相关。近来的研究发现内皮型一氧化氮合酶（eNOS）基因7外显子中的G894T突变和CHD的发病及其心肌梗塞（MI）风险有关。 二、研究目的： 我们应用聚合酶链反应（PCR）技术、限制性内切酶酶切分析和遗传学方法，测定了一组中国汉族正常人、CHD患者的eNOS基因G894T突变频率，并进行CHD经典危险因子和家族史调查，旨在探讨eNOS基因多态性和中国人CHD的发生及其MI风险的关系。 三、对象与方法： CHD患者106例，男74例、女32例，年龄65士10岁。病例符合1979年Vq4O诊断标准，58例经冠状动脉造影确诊。参照MONICA方案设计CHD登记调查表，对病例进行经典危险因子及心血管家族史调查。正常人 108例，男68例、女40例，年龄64士8岁。选自人群健康体检的随机个体。研究人群个体之间无血缘关系。 所有受检者采血前禁食12小时，次晨空腹抽取静脉血。以常规方法测定血总胆固醇、血甘袖三酯。以快速盐析法从全血中提取DNA。参考 Hingorani AD等设计引物，经 PCR扩增，207hp的 PCR产物用 Ban 11酶消化。当 894位等位基因为 G时，有单个的 Ban 11酶切位点。而当 894位等位基因为 T时，没有Banff酶切位点。PCR产物用 Banff酶消化后，GG基因型产生 125hp和 82hp两种片断，GT基因型产生 207hp、125hp和 82hp三种片断，TT基因型只有一个片断。酶切产物用3％琼脂糖凝胶电泳，EB染色，紫外摄影，检测eNOS基因多态性。用基因计数法计算各组病例和正常人的基因型和等位基因频率。资料用 SPSS．0软件包作统计学分析。 四、结果： 1、经计算，正常对照组、CHD及其MI、AP亚组的基因型分布均符合 Hardyweinbeyg平衡。 2。中国汉族正常人群GG。GT。TT基因型频率分别为0．907、0．093和 0000；G、T等位基因频率分别为 0．954和 0刀46。 3、我们假设eNOS基因为显性遗传，其GT＋TT基因型频率与GG型相 比，CHD组、MI亚组均显著高于正常人（P均＜0．05），AP亚组与 正常人相比无显著差异（P＞0刀5）。上述各组T、G等位基因频率相比， ＊＊D组及＊1亚组均极显著高于正常人尸均＜0刀1），＊P亚组显著高 2 于正常人o＜0刀5）。 4、CHD患者MI组与AP组之间，eNOS基因型及等位基因频率差异 均无显著性o均＞0刀5）。 5、冠状动脉造影确诊的CHD患者eNOS基因GT＋TT基因型频率与GG 型相比，在单支、双支和多支血管病变组之间，差异均无显著性P 均＞0．05）。三组之间等位基因频率均无显著差异o均＞0刀5）。 6、CHD组eNOS基因G894T突变的基因型与CHD危险因子比较，可 见eNOS基因型GT＋TT与GG相比，和各危险因于之间无显著关系 p均＞0．05）。 7。CHD患者中有家族史组 GT＋TT型频率为 0．182，无家族史组 GT＋TT 型频率为0．219，与＊G相比，差异无显著性（P＞0．05）。两组等位基 因频率差异无显著性o＞O．05）。 五、结论： 1、中国汉族正常人eNOS基因G894T突变GG、GT、TT基因型频率分 别为0．907、0．093和0刀00刃和T等位基因频率分别为0．954和0．046。 2、中国汉族正常人GT＋TT基因型频率、T等位基因频率显著低于白种 人和日本人。 3、CNOS基因G894T突变与中国汉族人群CHD的发生有关。 4、eNOS基因G894T突变与CHD经典危险因子无关。eNOS基因G894T 突变可能是中国人CHD的一个独立的危险因于。 5、eNOS基因G894T突变与CHD病变血管支数无关。 6、eNOS基因G894T突变与中国人CHD患者心血管病家族史无关。更多还原

【Abstract】 Background.Epidemiologic studies have shown that hyperlipidemia, hypertension, cigarette smoking, diabetes mellitus, obesity and family history of cardiovascular disease are the risk factors for coronary heart disease (CHD). Although the accumulation of coronary risk factors often leads to CHD, however, there is a population of patients without such classic risk factors who develop coronary artery disease. CHD may arise from interactions between environmental and genetic factors in general, but the precise mechanism by which coronary heart disease occurs remains to be elucidated. This study tested the hypothesis that a missense Glu298Asp variant within exon 7 of the endothelial nitric oxide synthase (eNOS) gene may be a potent risk for CHD in Chinese population. Methods.To examine the distribution of the missense Glu298Asp variant in exon on 7 of the eNOS gene, polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) analysis was performed in the 106 unrelated CHD patients (74 men and 32 women, mean age 65?0 years) and 108 unrelated normal subjects (68 men and 40 women, mean age 64? years). AH patients met thediagnostic criteria for ischemic heart disease by the World Health Organization (WHO), among which 58 ones were confirmed by coronary angiography. There was no relationship between individuals in the study population.Peripheral venous blood samples were collected after a 12-hour overnight fast. Serum total cholesterol (TC) and triglyceride (TG) were measured by conventional methods. Genomic DNA was extracted from whole blood by rapid salting out method. Using the primers described by Hingorani AD,et al ,we performed polymerase chain reaction (PCR) amplification. The 207bp PCR product for exon 7 was digested with direct restriction enzyme, Ban II. A single Ban II site was present in the wild type allele (G at 894), whereas no Ban II site was found in the mutant allele (T at 894). Therefore, treatment of the wild type (GG) with Ban II yielded 125bp and 82bp fragments. In the case of the heterozygous mutant, digestion with Ban II resulted in three fragments of 207,125 and 82bp in size. And a single 207bp fragment was observed upon in the homozygous variant (TT) with Ban II. These products were electrophoresed on 3% agarose gels, and DNA was visualized by ethidium bromide straining.The gene counting method was used to estimate the allele and genotype frequencies in patients group and normal subjects. StatisticVanalyses were performed with the SPSS 10.0. Values were considered statisticallysignificant at PO.05.Results.1. The genotype distribution was compatible with the Hardy-Weinberg equilibrium in the CHD, as well as MI and angina pectoris (AP) subgroups.2. Among the normal subjects, the frequencies of the eNOS/GG, GT and TTgenotypes were 0.907, 0.093 and 0.000, respectively. The G and T allele frequencies were 0.954 and 0.046.3. We assumed the effects of the T allele to be dominant (GT and TT combined vs GG), the eNOS genotypes distribution in CHD patients, as well as MI subgroup were significantly different from the normal subjects (P<0.05). The frequencies of T allele in CHD ,MI and AP subgroups were significantly higher than that in the normal subjects (PO.05), respectively. The eNOS genotypes distribution or the frequencies of T allele had no difference between MI and AP subgroups.4. We found no difference between the eNOS variant (include the distribution of genotypes and the frequencies of T allele ) and the number of diseased vessels in the CHD group (P>0.05).5. There were no differences among genotypes in terms of years, sex, body mass index, systolic blood pressure, diastolic blood pressure, TC, TG. The prevalences of hyperlipidemia, hypertension (HT), cigrette smoking, diabetes mellitus and obesity, were similar in the eNOS genotypes (P>0.05).6. The eNOS genotype distribution has no difference between CHD patients with and without family history of cardiovascular disease (P>0.05).7. We found significant differences更多还原