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基质金属蛋白酶-1基因多态性与特发性肺纤维化相关性的研究

发布时间:2018-05-23 14:34

  本文选题:特发性肺纤维化 + 基质金属蛋白酶-1 ; 参考:《河北医科大学》2012年硕士论文


【摘要】:特发性肺纤维化是一种慢性致命性、以肺部组织异常重塑为特征的间质性肺部疾病。其病因不明,目前认为是环境因素与多种遗传因素相互作用可能参与其疾病的发展。基质金属蛋白酶-1其主要作用是降解细胞外基质成份,认为其在肺纤维化的形成中起了重要作用。近年来研究显示MMP-1具有基因多态性现象,其与哮喘、慢性阻塞型肺疾病、肺结核等多种肺疾病之间的易感性已有报道。而国内尚无关于MMP-1基因多态性与特发性肺纤维化的研究报道。本实验旨在通过研究基质金属蛋白酶-1(Matrix metalloproteinase-1)启动子区-1607位点1G/2G单核苷酸基因多态性(single nucleotide polymorphism,,SNP)与中国河北地区汉族特发性肺纤维化的相关性,并探测特发性肺纤维化患者血清学MMP-1和TIMP-1的蛋白含量,探讨中国河北地区汉族特发性肺纤维化患者的遗传易感因素、发病机制及该位点基因多态性在不同种族特发性肺纤维化患者易感性方面的差异。 方法:采用基于人群的病例-对照研究方法,参考2002年中华医学会颁布的《特发性肺纤维化诊断和治疗指南(草案)》中的诊断标准,选取临床上诊断为特发性肺纤维化(IPF)汉族患者84例,其中男性51例,女性33例,年龄43岁-88岁,平均(66.7±9.5)岁,且除外继发性肺纤维化、急性感染、肿瘤、栓塞、心衰、控制不良的糖尿病、消化性溃疡、严重骨质疏松、结核病患者。对照组:选取汉族健康体检人群100例,其中男性53例,女性47例,年龄40岁-73岁,平均年龄(50.2±7.5)岁,且既往无变态反应性疾病病史,近期无感染,无免疫抑制剂应用史。采集IPF患者及健康对照组外周静脉血5ml,其中2mlEDTA抗凝,以消化-苯酚-氯仿有机溶剂法和树脂型TM基因组DNA提纯试剂盒提取外周血白细胞DNA,利用限制性内切酶ALUI,聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术结合琼脂糖凝胶电泳的方法,检测IPF患者和健康对照者MMP-1基因启动子区-1607(1G/2G)多态性位点的基因型和等位基因分布,分析两组人群MMP-l基因型和等位基因频率的差异。余3ml静脉血促凝剂促凝,采用双抗夹心酶联免疫吸附法(ELISA)测定IPF患者和健康对照组MMP-1和TIMP-1的血清蛋白表达水平。 结果: 1MMP-1位点基因多态性分析 1.1MMP-1-1607的基因型 本实验检测得到三种基因型:1G/1G型、1G/2G型及2G/2G型;其中1G/1G型为野生型,1G/2G型及2G/2G型为突变型。IPF组与健康对照组均符合Hardy-Weinberg遗传平衡性检验(P0.05),两组之间具有良好的可比性。 1.2MMP-1-16071G/2G各基因型在两组间的分布情况 IPF组84例中检测出1G/1G基因型12例(14.3%),1G/2G基因型30例(35.7%),2G/2G基因型42例(50%)。健康对照组100例中检测出1G/1G基因型11例(11%),1G/2G基因型42例(42%),2G/2G基因型47例(47%)。MMP-1-1607位点各基因型在两组间的分布频率比较无明显统计学差异(χ2=0.940, P0.05)。 1.3MMP-1-1607位点1G/2G各等位基因频率在两组间的分布情况 IPF组84例中1G等位基因频率为32.1%,2G等位基因频率为67.9%,健康对照组100例中1G等位基因频率为32%,2G等位基因频率为68%。MMP-1-1607位点的各等位基因在两组间的分布频率比较无显著统计学差异性(χ2=0.001, P0.05)。 2血清MMP-1,TIMP-1蛋白浓度的变化 2.1血清MMP-1蛋白浓度变化: IPF患者与健康对照组MMP-1血清蛋白浓度分别为:374.66±54.18ng/ml,97.45±20.39ng/ml。IPF患者血清MMP-1蛋白浓度明显高于健康对照组,两组比较有统计学意义(P0.05)。 2.2血清TIMP-1蛋白浓度变化: IPF组与健康对照组TIMP-1血清蛋白浓度分别为:111.10±26.70ng/ml,40.75±11.46ng/ml。IPF患者血清TIMP-1蛋白浓度较健康对照组明显升高,两组比较有统计学意义(P0.05)。 2.3用MMP-1/TIMP-1蛋白浓度得出两者的比值 IPF组与健康对照组MMP-1/TIMP-1比值分别为:3.39±1.06,2.72±1.00,两组比较有统计学意义(P0.05)。 结论: 1.结合本研究推测MMP-1-1607位点的1G/2G基因多态性与中国河北地区汉族人群特发性肺间质纤维化的易感性无明显相关性,结合对其它地区、种族人群的研究,说明该位点基因多态性与IPF的易感性存在种族、地区的差异。 2. IPF患者肺纤维化的发生与MMP-1,TIMP-1水平升高可能有关,MMP-1和TIMP-1可能参与IPF疾病的发生发展。
[Abstract]:Idiopathic pulmonary fibrosis is a chronic and fatal interstitial lung disease characterized by abnormal remodeling of the lung tissue. Its etiology is unknown. It is considered that environmental factors interact with a variety of genetic factors and may participate in the development of its disease. Matrix metalloproteinase -1 is mainly used to degrade extracellular matrix components and consider it in the lung. There has been an important role in the formation of fibrosis. In recent years, studies have shown that MMP-1 has genetic polymorphisms, and its susceptibility to multiple lung diseases such as asthma, chronic obstructive pulmonary disease, tuberculosis and other lung diseases has been reported. However, there is no report on the polymorphism of MMP-1 gene and idiopathic pulmonary fibrosis in China. The aim of this experiment is to study the study. The correlation between 1G/2G single nucleotide polymorphism (single nucleotide polymorphism, SNP) in the promoter region of the matrix metalloproteinase -1 (Matrix metalloproteinase-1) and the Han idiopathic pulmonary fibrosis in Hebei, China, and detection of the protein content of MMP-1 and TIMP-1 in the blood of patients with idiopathic pulmonary fibrosis and the exploration of Hebei land in China Genetic susceptibility factors, pathogenesis and genetic polymorphisms of this loci in patients with idiopathic pulmonary fibrosis in different ethnic groups in patients with idiopathic pulmonary fibrosis.
Methods: a population based case control study was used, referring to the diagnostic criteria for the diagnosis and treatment of idiopathic pulmonary fibrosis (Draft) issued by the Chinese Medical Association of 2002, and 84 cases of Han patients diagnosed as idiopathic pulmonary fibrosis (IPF) were selected clinically, including 51 male, 33 female, 43 years old, and an average of (66.7 + 9.5). With the exception of secondary pulmonary fibrosis, acute infection, tumor, embolism, heart failure, poor control of diabetes, peptic ulcers, severe osteoporosis, and tuberculosis patients. Control group: 100 cases of Han health examination were selected, of which 53 were male, 47 women, age 40 -73 years, average age (50.2 + 7.5) years, and no previous allergy disease. History of disease, no infection, no history of immunosuppressant application. Collection of peripheral venous blood 5ml in IPF patients and healthy control group, including 2mlEDTA anticoagulant, extraction of DNA from peripheral blood leukocyte by digestion phenol chloroform organic solvent method and resin type TM genomic DNA purification kit, using restrictive endonuclease ALUI, polymerase chain reaction restriction fragment length PCR-RFLP technique combined with agarose gel electrophoresis to detect the genotype and allele distribution of the -1607 (1G/2G) polymorphic loci in the promoter region of the MMP-1 gene of IPF and healthy controls. The difference between the MMP-l genotypes and allele frequencies of the two groups was analyzed. The coagulant promoting coagulant in the venous blood of the two groups was used and the double resistant sandwich enzyme was used. Serum protein levels of MMP-1 and TIMP-1 in IPF patients and healthy controls were measured by ELISA.
Result锛

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