本文關(guān)鍵詞：ADAM33及T細(xì)胞因子在維吾爾族COPD患者氣道重塑中的調(diào)控作用研究　出處：《新疆醫(yī)科大學(xué)》2014年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： COPD ADAM33 Meta分析 原代肺成纖維細(xì)胞 細(xì)胞因子

【摘要】：目的：1)通過Meta分析ADAM33基因T1(rs2280091), S1(rs3918396)和S2(rs528557)位點(diǎn)與慢性阻塞性肺病的易感性之間的關(guān)系；2)對(duì)維吾爾族慢性阻塞性肺疾病患者及正常維吾爾族原代肺成纖維細(xì)胞進(jìn)行體外培養(yǎng)、鑒定；3)通過測(cè)定維吾爾族慢性阻塞性肺病患者及正常維吾爾族肺成纖維細(xì)胞中ADAM33蛋白及基因的表達(dá),探討是否維吾爾族慢性阻塞性肺病患者原代肺成纖維細(xì)胞中ADAM33存在高表達(dá)；4)探討是否Th1/Th2細(xì)胞因子通過調(diào)控ADAM33的表達(dá),作用在肺成纖維細(xì)胞,從而影響氣道重塑,進(jìn)而抑制或促進(jìn)COPD的發(fā)生發(fā)展,為進(jìn)一步研究ADAM33及T細(xì)胞因子對(duì)氣道重塑的作用機(jī)制,并幫助開發(fā)慢性阻塞性肺病的治療方法應(yīng)用于臨床實(shí)踐中提供理論依據(jù)。方法：1)收集Pubmed數(shù)據(jù)庫(kù),Embase數(shù)據(jù)庫(kù),中國(guó)國(guó)家知識(shí)基礎(chǔ)設(shè)施數(shù)據(jù)庫(kù)(CNKI)和萬方數(shù)據(jù)庫(kù)中2012年9月5日之前發(fā)表的有關(guān)ADAM33基因多態(tài)性與慢性阻塞性肺病的易感性之間關(guān)聯(lián)的文獻(xiàn),兩個(gè)獨(dú)立工作人員仔細(xì)的評(píng)估所有的研究,明確復(fù)合納入標(biāo)準(zhǔn)的文獻(xiàn),提取每項(xiàng)研究中的下列信息：第一作者的姓名,出版年份,種族,病例和對(duì)照數(shù),慢性阻塞性肺病的定義,病例組和對(duì)照組的基因型分布,使用METAGEN(STATA12.0)和Revman5.0軟件進(jìn)行統(tǒng)計(jì)分析；2)由新疆醫(yī)科大學(xué)第一附屬醫(yī)院胸外科提供因肺癌進(jìn)行手術(shù)切除標(biāo)本的正常肺組織,采用貼壁法分離維吾爾族慢性阻塞性肺病患者及正常維吾爾族肺成纖維細(xì)胞,進(jìn)行體外培養(yǎng),采用共聚焦顯微鏡進(jìn)行鑒定；3)采用RT-PCR、Western blot方法測(cè)定維吾爾族慢性阻塞性肺病患者及正常維吾爾族肺成纖維細(xì)胞中ADAM33的水平；4)在正常維吾爾族肺成纖維細(xì)胞培養(yǎng)基中加入相同濃度的細(xì)胞因子(IL-4, IL-13, INF-γ)(100ng/ml),加入細(xì)胞因子前血清饑餓24小時(shí),刺激不同的時(shí)間后(0h、6h、12h、24h、48h)收集細(xì)胞,采用RT-PCR. Westernblot測(cè)定肺成纖維細(xì)胞中ADAM33的水平；在正常維吾爾族肺成纖維細(xì)胞培養(yǎng)基中加入不同濃度的細(xì)胞因子(IL-4, IL-13, INF-γ)(0,1ng/ml、10ng/ml、100ng/ml),加入細(xì)胞因子前血清饑餓24小時(shí),刺激24h后收集細(xì)胞,采用RT-PCR、Western blot測(cè)定肺成纖維細(xì)胞中ADAM33的水平。結(jié)果：1)Meta分析共納入10項(xiàng)病例對(duì)照研究,其中包括慢性阻塞性肺疾病患者2139例和健康對(duì)照組3765例。結(jié)果表明：S2(rs528557)和T1(rs2280091)并沒有增加或降低慢性阻塞性肺病的易感性。S1(rs3918396)(GG+AG vs. AA)與亞洲人群慢性阻塞性肺病易感性顯著相關(guān)：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)波形蛋白免疫熒光染色共聚焦顯像結(jié)果提示：從肺組織塊原代分離培養(yǎng)的細(xì)胞中99%以上表達(dá)波形蛋白；3)通過熒光定量RT-PCR分析ADAM33基因在維吾爾族COPD患者肺原代成纖維細(xì)胞和正常肺成纖維細(xì)胞中的表達(dá)模式,P=0.0420.05,差異有統(tǒng)計(jì)學(xué)意義。同時(shí),利用Western blot技術(shù)檢測(cè)ADAM33蛋白在維吾爾族COPD患者肺原代成纖維細(xì)胞和維吾爾族正常肺成纖維細(xì)胞中的表達(dá)情況,P=0.0110.05,差異有統(tǒng)計(jì)學(xué)意義；4)隨著INF-y刺激時(shí)間的增加(0h、6h、12h、24h、48h),ADAM33基因及蛋白的表達(dá)量顯著降低,INF-γ刺激時(shí)間與2-(△△Ct呈負(fù)相關(guān)(r=-0.399,P=0.039), INF-γ刺激時(shí)間與蛋白表達(dá)呈負(fù)相關(guān)(r=-0.268,P=0.048)；同樣隨著INF-γ刺激濃度的增加(0ng/ml,1ng/ml,10ng/ml,100ng/ml), ADAM33基因及蛋白的表達(dá)量顯著降低,INF-γ刺激濃度與2-(△△Ct)呈負(fù)相關(guān)(r=-0.492, P=0.027),INF-γ刺激時(shí)間與蛋白表達(dá)呈負(fù)相關(guān)(r=-0.558,P=0.011)。隨著IL-4刺激時(shí)間的增加(Oh、6h、12h、24h、48h), ADAM31基因及蛋白的表達(dá)量顯著增高,IL-4刺激時(shí)間與2-(△△Ct)呈正相關(guān)(r=0.888, P=0.041), IL-4刺激時(shí)間與蛋白表達(dá)呈正相關(guān)(r=0.703,P=0.038)；同樣隨著IL-4刺激濃度的增加(0ng/ml,1ng/ml,10ng/ml,100ng/ml), ADAM33基因及蛋白的表達(dá)量顯著增高,IL-4刺激濃度與2-(△△Ct)呈正相關(guān)(r=0.383, P=0.004), IL-4刺激時(shí)間與蛋白表達(dá)呈正相關(guān)(r=0.508, P=0.003)。IL-13;刺激時(shí)間與2-△△Ct無相關(guān)性(r=-0.254,P=0.108),亦與IOD值無相關(guān)(r=-0.189, P=0.453); IL-13刺激濃度與2-(△△Ct)無相關(guān)性(r=-0.535,P=0.37)；亦與IOD值無相關(guān)性(r=-0.351,P=0.08)。結(jié)論：1)通過對(duì)數(shù)據(jù)庫(kù)中與COPD及ADAM33相關(guān)的研究進(jìn)行Meta分析,研究了ADAM33和慢性阻塞性肺病的易感性之間的關(guān)聯(lián)。我們的研究結(jié)果表明：S2(rs528557), T1(rs2280091)純合子攜帶者并沒有增加或減少慢性阻塞性肺病的風(fēng)險(xiǎn)；在亞洲人群S1(rs3918396)(GG+AG vs.AA)與慢性阻塞性肺病顯著相關(guān)；2)分離和培養(yǎng)出維吾爾族COPD患者以及正常維吾爾族肺原代成纖維細(xì)胞；3)ADAM33在維吾爾族COPD患者原代肺成纖維細(xì)胞中的表達(dá)水平較正常維吾爾族肺成纖維細(xì)胞中的表達(dá)明顯；4) IFN-γ可能通過抑制ADAM33的表達(dá),作用在肺成纖維細(xì)胞；IL-4則可能相反,通過增加ADAM33的表達(dá),作用在肺成纖維細(xì)胞,從而影響氣道重塑,進(jìn)而抑制或促進(jìn)COPD的發(fā)生發(fā)展,為進(jìn)一步研究ADAM33及T細(xì)胞因子對(duì)氣道重塑的作用機(jī)制,并幫助開發(fā)慢性阻塞性肺病的治療方法應(yīng)用于臨床實(shí)踐中提供理論依據(jù)。
[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
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R563.9

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李靜;李彬彬;尹磊;陳會(huì)良;劉德義;;沙參麥冬湯對(duì)慢支模型大鼠肺臟抗氧化能力及其組織結(jié)構(gòu)的影響[J];安徽科技學(xué)院學(xué)報(bào);2013年03期

2 韋袞政;和菊花;劉濤;錢銳;張馨予;儲(chǔ)麗英;祁向榮;;肺脹陽虛水泛證動(dòng)物模型的建立方法[J];環(huán)球中醫(yī)藥;2011年04期

3 姚寶林;楊麗蕓;趙靜;陳三班;宋躍朋;;補(bǔ)氣活血方對(duì)慢性支氣管炎肺氣虛證大鼠肺組織細(xì)胞凋亡的影響[J];解放軍醫(yī)藥雜志;2014年06期

4 李慶云,黃紹光,周同,程齊儉,吳華成,許海敏,李敏,鄧偉吾;吸煙致大鼠慢性支氣管炎形成過程氣道上皮ICAM-1表達(dá)的研究[J];中國(guó)微循環(huán);2005年05期

5 朱偉群;劉漢勝;;養(yǎng)陰清肺糖漿對(duì)煙霧引起的慢性支氣管炎大鼠炎癥細(xì)胞及SOD、MDA、NO的影響[J];中藥材;2006年03期

6 唐宇姣;楊宇;惠毅;陳小兵;唐洪屈;王寶家;周新穎;;“肺病及腸”動(dòng)物模型建立的探索[J];中華中醫(yī)藥學(xué)刊;2012年08期

7 黃紅坤;黃小紅;劉盛來;黃小瓊;嚴(yán)家榮;王諾;趙偉健;王剛;;氣管炎丸治療慢性支氣管炎作用機(jī)制的研究[J];中國(guó)醫(yī)藥指南;2013年13期

8 李靜;李彬彬;尹磊;陳會(huì)良;劉德義;;沙參麥冬湯對(duì)慢性支氣管炎模型大鼠抗氧化能力的影響[J];中醫(yī)雜志;2013年17期

9 周運(yùn)海;楊洋;;何氏加味定喘湯對(duì)慢性支氣管炎大鼠內(nèi)皮素-1及肺組織形態(tài)學(xué)的影響[J];吉林中醫(yī)藥;2013年10期

10 陳章;耿延?xùn)|;歐陽剛;;沙茯膠囊對(duì)小鼠慢性支氣管炎模型的藥理學(xué)作用[J];西部醫(yī)學(xué);2014年06期

相關(guān)博士學(xué)位論文 前2條

1 姜偉洲;基于中醫(yī)理論的肺陰虧虛、寒邪犯肺證動(dòng)物模型的建立及相關(guān)理論的研究[D];山東中醫(yī)藥大學(xué);2010年

2 楊東斌;吸煙對(duì)重型顱腦損傷伴發(fā)肺損傷的影響及穿心蓮內(nèi)酯在吸煙誘發(fā)肺損傷中的保護(hù)機(jī)制研究[D];鄭州大學(xué);2013年

相關(guān)碩士學(xué)位論文 前5條

1 朱雪;基于中醫(yī)理論的肺實(shí)熱證動(dòng)物模型的研究[D];山東中醫(yī)藥大學(xué);2010年

2 王震;高遷移率族蛋白B1在香煙暴露大鼠模型中的表達(dá)及其意義[D];安徽醫(yī)科大學(xué);2011年

3 歐陽金生;脂多糖—煙霧誘導(dǎo)慢性支氣管炎大鼠模型水通道蛋白5表達(dá)與腫瘤壞死因子α、白介素13、白三烯B_4的變化[D];福建醫(yī)科大學(xué);2006年

4 吳琴琴;ADAM33基因在慢性阻塞性肺疾病中的表達(dá)及意義[D];貴陽中醫(yī)學(xué)院;2009年

5 唐宇姣;從肺病模型大鼠病理改變及相關(guān)活性物質(zhì)SP、VIP、INOS含量變化探討“肺病及腸”病理傳變機(jī)制[D];成都中醫(yī)藥大學(xué);2012年

，

本文編號(hào)：1340334