ADAM33及T细胞因子在维吾尔族COPD患者气道重塑中的调控作用研究

发布时间：2017-12-27 05:05

本文关键词：ADAM33及T细胞因子在维吾尔族COPD患者气道重塑中的调控作用研究　出处：《新疆医科大学》2014年博士论文　论文类型：学位论文

【摘要】：目的：1)通过Meta分析ADAM33基因T1(rs2280091), S1(rs3918396)和S2(rs528557)位点与慢性阻塞性肺病的易感性之间的关系；2)对维吾尔族慢性阻塞性肺疾病患者及正常维吾尔族原代肺成纤维细胞进行体外培养、鉴定；3)通过测定维吾尔族慢性阻塞性肺病患者及正常维吾尔族肺成纤维细胞中ADAM33蛋白及基因的表达,探讨是否维吾尔族慢性阻塞性肺病患者原代肺成纤维细胞中ADAM33存在高表达；4)探讨是否Th1/Th2细胞因子通过调控ADAM33的表达,作用在肺成纤维细胞,从而影响气道重塑,进而抑制或促进COPD的发生发展,为进一步研究ADAM33及T细胞因子对气道重塑的作用机制,并帮助开发慢性阻塞性肺病的治疗方法应用于临床实践中提供理论依据。方法：1)收集Pubmed数据库,Embase数据库,中国国家知识基础设施数据库(CNKI)和万方数据库中2012年9月5日之前发表的有关ADAM33基因多态性与慢性阻塞性肺病的易感性之间关联的文献,两个独立工作人员仔细的评估所有的研究,明确复合纳入标准的文献,提取每项研究中的下列信息：第一作者的姓名,出版年份,种族,病例和对照数,慢性阻塞性肺病的定义,病例组和对照组的基因型分布,使用METAGEN(STATA12.0)和Revman5.0软件进行统计分析；2)由新疆医科大学第一附属医院胸外科提供因肺癌进行手术切除标本的正常肺组织,采用贴壁法分离维吾尔族慢性阻塞性肺病患者及正常维吾尔族肺成纤维细胞,进行体外培养,采用共聚焦显微镜进行鉴定；3)采用RT-PCR、Western blot方法测定维吾尔族慢性阻塞性肺病患者及正常维吾尔族肺成纤维细胞中ADAM33的水平；4)在正常维吾尔族肺成纤维细胞培养基中加入相同浓度的细胞因子(IL-4, IL-13, INF-γ)(100ng/ml),加入细胞因子前血清饥饿24小时,刺激不同的时间后(0h、6h、12h、24h、48h)收集细胞,采用RT-PCR. Westernblot测定肺成纤维细胞中ADAM33的水平；在正常维吾尔族肺成纤维细胞培养基中加入不同浓度的细胞因子(IL-4, IL-13, INF-γ)(0,1ng/ml、10ng/ml、100ng/ml),加入细胞因子前血清饥饿24小时,刺激24h后收集细胞,采用RT-PCR、Western blot测定肺成纤维细胞中ADAM33的水平。结果：1)Meta分析共纳入10项病例对照研究,其中包括慢性阻塞性肺疾病患者2139例和健康对照组3765例。结果表明：S2(rs528557)和T1(rs2280091)并没有增加或降低慢性阻塞性肺病的易感性。S1(rs3918396)(GG+AG vs. AA)与亚洲人群慢性阻塞性肺病易感性显著相关：ORtotal=1.27[95%confidence interval (CI)1.03-1.56, P=0.03], ORAsian=1.44(95%CI1.13-1.83, P=0.003);2)波形蛋白免疫荧光染色共聚焦显像结果提示：从肺组织块原代分离培养的细胞中99%以上表达波形蛋白；3)通过荧光定量RT-PCR分析ADAM33基因在维吾尔族COPD患者肺原代成纤维细胞和正常肺成纤维细胞中的表达模式,P=0.0420.05,差异有统计学意义。同时,利用Western blot技术检测ADAM33蛋白在维吾尔族COPD患者肺原代成纤维细胞和维吾尔族正常肺成纤维细胞中的表达情况,P=0.0110.05,差异有统计学意义；4)随着INF-y刺激时间的增加(0h、6h、12h、24h、48h),ADAM33基因及蛋白的表达量显著降低,INF-γ刺激时间与2-(△△Ct呈负相关(r=-0.399,P=0.039), INF-γ刺激时间与蛋白表达呈负相关(r=-0.268,P=0.048)；同样随着INF-γ刺激浓度的增加(0ng/ml,1ng/ml,10ng/ml,100ng/ml), ADAM33基因及蛋白的表达量显著降低,INF-γ刺激浓度与2-(△△Ct)呈负相关(r=-0.492, P=0.027),INF-γ刺激时间与蛋白表达呈负相关(r=-0.558,P=0.011)。随着IL-4刺激时间的增加(Oh、6h、12h、24h、48h), ADAM31基因及蛋白的表达量显著增高,IL-4刺激时间与2-(△△Ct)呈正相关(r=0.888, P=0.041), IL-4刺激时间与蛋白表达呈正相关(r=0.703,P=0.038)；同样随着IL-4刺激浓度的增加(0ng/ml,1ng/ml,10ng/ml,100ng/ml), ADAM33基因及蛋白的表达量显著增高,IL-4刺激浓度与2-(△△Ct)呈正相关(r=0.383, P=0.004), IL-4刺激时间与蛋白表达呈正相关(r=0.508, P=0.003)。IL-13;刺激时间与2-△△Ct无相关性(r=-0.254,P=0.108),亦与IOD值无相关(r=-0.189, P=0.453); IL-13刺激浓度与2-(△△Ct)无相关性(r=-0.535,P=0.37)；亦与IOD值无相关性(r=-0.351,P=0.08)。结论：1)通过对数据库中与COPD及ADAM33相关的研究进行Meta分析,研究了ADAM33和慢性阻塞性肺病的易感性之间的关联。我们的研究结果表明：S2(rs528557), T1(rs2280091)纯合子携带者并没有增加或减少慢性阻塞性肺病的风险；在亚洲人群S1(rs3918396)(GG+AG vs.AA)与慢性阻塞性肺病显著相关；2)分离和培养出维吾尔族COPD患者以及正常维吾尔族肺原代成纤维细胞；3)ADAM33在维吾尔族COPD患者原代肺成纤维细胞中的表达水平较正常维吾尔族肺成纤维细胞中的表达明显；4) IFN-γ可能通过抑制ADAM33的表达,作用在肺成纤维细胞；IL-4则可能相反,通过增加ADAM33的表达,作用在肺成纤维细胞,从而影响气道重塑,进而抑制或促进COPD的发生发展,为进一步研究ADAM33及T细胞因子对气道重塑的作用机制,并帮助开发慢性阻塞性肺病的治疗方法应用于临床实践中提供理论依据。
[Abstract]:Objective: 1) by the analysis of Meta ADAM33 gene T1 (rs2280091), S1 (rs3918396) and S2 (rs528557) the relationship between susceptibility loci and chronic obstructive pulmonary disease; 2) of Uygur patients with chronic obstructive pulmonary disease and normal Uygur primary lung fibroblasts were cultured in vitro and identified by 3); Determination of Uygur patients with chronic obstructive pulmonary disease and normal Uygur lung ADAM33 protein and gene expression in fiber cells, to investigate whether the Uygur patients with chronic obstructive pulmonary disease in primary lung fibroblast ADAM33 in the presence of high expression; 4) to investigate whether Th1/Th2 cells by regulating the expression of ADAM33 in lung fibroblasts, thus. Effect of airway remodeling, and inhibit or promote the occurrence and development of COPD, ADAM33 and T for further study on the mechanism of cellular factors on airway remodeling, and help develop chronic obstructive The treatment of lung disease is used in clinical practice to provide theoretical basis. Methods: 1) collected from the Pubmed database, Embase database, Chinese national knowledge infrastructure database (CNKI) related literature published before September 5, 2012 and the susceptibility of Wanfang database on ADAM33 gene polymorphism and chronic obstructive pulmonary disease, all the study assessed two independent staff carefully, clear composite inclusion criteria of literature the following information extraction, in each of the studies: the first author's name, year of publication, race, number of cases and controls, the definition of chronic obstructive pulmonary disease, genotype distribution in the case group and the control group (STATA12.0), using METAGEN and Revman5.0 software for statistical analysis; 2) from the Department of thoracic surgery of the First Affiliated Hospital of Xinjiang Medical University from lung cancer normal lung tissues of surgical specimens were isolated by adherent Uygur patients with chronic obstructive pulmonary disease and normal lung Uygur Fibroblasts were cultured in vitro and identified by confocal microscope; 3) determination of Uygur patients with chronic obstructive pulmonary disease and normal Uygur lung fibroblasts ADAM33 level by RT-PCR, Western blot; 4) in normal cells with the same concentration of Uygur factor based in lung fibroblasts (IL-4, IL-13, INF- gamma) (100ng/ml), adding the cytokines before serum starvation for 24 hours, after stimulation in different time (0h, 6h, 12h, 24h, 48h) were collected for determination of lung fibroblast ADAM33 in RT-PCR. level by Westernblot in normal cultured cells; Uygur factor with different concentration of radicals in lung fibroblasts (IL-4, IL-13, INF-) (0,1ng/ml, 10ng/ml, 100ng/ml), adding the cytokines before serum starvation for 24 hours, stimulation of 24h cells were collected after determination of lung by RT-PCR, Western blot The level of ADAM33 in fibrous cells. Results: 1) a total of 10 case control studies were included in Meta analysis, including 2139 patients with chronic obstructive pulmonary disease and 3765 cases of healthy control. The results showed that S2 (rs528557) and T1 (rs2280091) did not increase or decrease the susceptibility to chronic obstructive pulmonary disease. S1 (rs3918396) (GG+AG vs. AA) were significantly associated with susceptibility to chronic obstructive pulmonary disease in Asian population: ORtotal=1.27[95%confidence interval (CI) 1.03-1.56, P=0.03], ORAsian=1.44 (95%CI1.13-1.83, P=0.003); 2) vimentin immunofluorescence confocal imaging showed that more than 99% blocks from primary lung tissue cultured cells expressed vimentin; 3) by fluorescence quantitative RT-PCR analysis of P=0.0420.05 ADAM33 gene in Uygur COPD patients with pulmonary primary fibroblasts and normal lung fibroblast expression pattern, the difference was statistically significant. At the same time, using the Western blot technique to detect the expression of ADAM33 in Uygur COPD patients with pulmonary primary fibroblasts and Uygur normal lung expression, cells in the P=0.0110.05, the difference was statistically significant; 4) with the increase of INF-y stimulation time (0h, 6h, 12h, 24h, 48h), the expression of ADAM33 gene and protein the decreased INF- and 2- (gamma stimulation time was negatively related to delta Ct (r=-0.399, P=0.039), INF- gamma stimulation time and protein expression was negatively correlated (r=-0.268, P=0.048); the same with the increase of INF- gamma concentration (0ng/ml, 1ng/ml, 10ng/ml, 100ng/ml), the expression of ADAM33 gene and protein significantly decreased, INF- concentration and 2- stimulation (gamma delta Ct) negative correlation (r=-0.492, P=0.027), INF- gamma stimulation time and protein expression was negatively correlated (r=-0.558, P=0.011). With the increase of IL-4 stimulation time (Oh, 6h, 12h, 24h, 48h), the expression of ADAM31 mRNA and protein was significantly increased, IL-4 stimulation time and 2- (delta Ct) were positively correlated (r=0.888, P=0.041), IL-4 protein expression was positively correlated with the stimulus duration (r=0.703, P=0.038); the same with the increase of IL-4 concentration (0ng/ml, 1ng/ml, 10ng/ml, 100ng/ml), the expression of ADAM33 mRNA and protein was significantly increased, IL-4 concentration and 2- (delta Ct) were positively correlated (r=0.383, P=0.004), IL-4 protein expression was positively correlated with the stimulus duration (r=0.508, P=0.003). IL-13; stimulation time and 2- Delta Ct (r=-0.254, P=0.108) had no correlation with IOD value, also no correlation (r=-0.189, P=0.453); IL-13 2- (stimulus concentration and delta Ct) no correlation (r=-0.535, P=0.37); also has no correlation with IOD value (r=-0.351, P=0.08). Conclusions: 1) the association between ADAM33 and the susceptibility to chronic obstructive pulmonary disease (COPD) was studied by Meta analysis of COPD and ADAM33 related studies in the database. Our results showed that S2 (rs528557), T1 (rs2280091) homozygous carriers do not increase or decrease the risk of chronic obstructive pulmonary disease; in Asian populations S1 (rs3918396) (GG+AG vs.AA) were significantly associated with chronic obstructive pulmonary disease; 2) isolated and cultured from Uygur nationality patients with COPD and normal Uygur primary lung fibroblasts; 3) ADAM33 in Uygur patients with COPD primary lung fibroblast expression level than in normal Uygur
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R563.9

【共引文献】