本文選題：慢性阻塞性肺疾病 + 痰液�。� 參考：《廣州醫(yī)學(xué)院》2012年碩士論文

【摘要】：【研究背景】： 慢性阻塞性肺疾病(Chronic Obstructive Pulmonary Disease, COPD)是世界范圍內(nèi)高發(fā)病率和高死亡率的慢性疾病之一，它以漸進(jìn)性不完全可逆的慢性氣流受限為特征，并與吸煙或吸入有害顆�；驓怏w引起的異常炎癥反應(yīng)有關(guān)。大量研究證明，氣道慢性炎癥是COPD發(fā)病的核心內(nèi)容，其形成機(jī)制十分復(fù)雜，涉及多種炎性細(xì)胞，在肺的不同部位有肺泡巨噬細(xì)胞、T淋巴細(xì)胞和中性粒細(xì)胞的增加，激活的炎癥細(xì)胞釋放各種炎性介質(zhì)如TNF-α，IL-8，從而損傷肺組織，促進(jìn)COPD的發(fā)生和發(fā)展。 粘蛋白MUC1（人類(lèi)用MUC1表示，，動(dòng)物用Muc1表示）是一種I型跨膜糖蛋白，分為細(xì)胞外區(qū)、跨膜區(qū)和胞內(nèi)區(qū)，主要分布在呼吸道、胃腸道、生殖道上皮細(xì)胞以及免疫相關(guān)細(xì)胞表面。它對(duì)正常上皮起潤(rùn)滑和保護(hù)作用，同時(shí)它的胞內(nèi)區(qū)還可以介導(dǎo)信號(hào)轉(zhuǎn)導(dǎo)和細(xì)胞粘附功能。近期研究表明不論是銅綠假單胞菌感染還是呼吸道合胞體病毒感染引起的急性炎癥中，都可以觀(guān)察到Muc1的表達(dá)量在感染后大量增加，而且這種表達(dá)增加與TNF-α、中性粒細(xì)胞彈性蛋白酶等的表達(dá)密切相關(guān)。從而提示Muc1是在感染和炎癥發(fā)生時(shí)起作用的重要內(nèi)源性分子。 但在出現(xiàn)肺部慢性炎癥的COPD患者中，肺中MUC1的表達(dá)水平如何，其表達(dá)水平是否也與炎癥因子水平相關(guān)，目前尚不清楚。 【研究目的】： 本實(shí)驗(yàn)研究了不同來(lái)源標(biāo)本，包括慢性阻塞性肺疾病（COPD）患者氣道粘膜和不同時(shí)期的痰液標(biāo)本，香煙煙霧誘導(dǎo)的COPD模型小鼠和細(xì)菌脂多糖誘導(dǎo)的急性炎癥動(dòng)物肺組織和支氣管肺泡灌洗液，以及利用香煙煙霧提取物和細(xì)菌脂多糖直接刺激的A549細(xì)胞中MUC1的表達(dá)改變情況。并分別檢測(cè)了COPD患者不同時(shí)期痰液中IL-8和TNF-α水平及COPD小鼠和急性炎癥小鼠支氣管肺泡灌洗液中TNF-α的水平。旨在揭示長(zhǎng)期煙霧刺激和急性細(xì)菌脂多糖刺激后小鼠肺內(nèi)Muc1的表達(dá)變化與TNF-α變化相關(guān)性，進(jìn)而探討COPD患者中MUC1的表達(dá)變化及其可能的機(jī)制，為COPD的發(fā)病機(jī)制提供新的理論基礎(chǔ)，并為COPD的預(yù)防和治療提供新的作用靶點(diǎn)。 【研究方法】： 本研究采用臨床病人標(biāo)本、動(dòng)物實(shí)驗(yàn)和細(xì)胞培養(yǎng)相結(jié)合的方法。（1）通過(guò)纖支鏡活檢留取COPD患者的氣道粘膜，采用免疫組化染色的方法來(lái)觀(guān)察MUC1的表達(dá)水平。分別收集COPD病人急性期和緩解期的痰液標(biāo)本，采用酶聯(lián)免疫吸附法（ELISA）檢測(cè)痰液上清中MUC1的量表達(dá)，同時(shí)檢測(cè)痰液中TNF-α和IL-8的水平。（2）利用單純熏煙6個(gè)月建立COPD小鼠模型，采用Western blot印跡法和ELISA檢測(cè)COPD模型小鼠肺內(nèi)Muc1表達(dá)水平，并檢測(cè)支氣管肺泡灌洗液中TNF-α的含量及細(xì)胞分類(lèi)計(jì)數(shù)。（3）采用脂多糖滴鼻致敏C57小鼠后建立急性炎癥動(dòng)物模型，分別收集12h、24h、48h、96h、1w后的肺組織和支氣管肺泡灌洗液，并分別采用Western blot和ELISA的方法檢測(cè)各時(shí)間段的肺內(nèi)Muc1的表達(dá)水平；同時(shí)檢測(cè)小鼠肺泡灌洗液中TNF-α的水平；（4）采用細(xì)胞培養(yǎng)的方法，分別用香煙煙霧提取物和脂多糖刺激A549細(xì)胞24h后，收集細(xì)胞蛋白，采用Western blot的方法檢測(cè)MUC1的表達(dá)水平。 【實(shí)驗(yàn)結(jié)果】： 一、COPD患者氣道粘膜上皮MUC1的表達(dá)增加 1、在COPD患者的氣道粘膜上皮MUC1免疫組化表達(dá)陽(yáng)性且較正常氣道粘膜的表達(dá)量增多。 2、 COPD患者急性期痰液上清中MUC1胞內(nèi)片段表達(dá)較緩解期明顯升高，且痰液上清IL-8水平、TNF-α水平均較緩解期升高，差異具有統(tǒng)計(jì)學(xué)意義（P0.05）；急性期痰液中白細(xì)胞總數(shù)、中性粒細(xì)胞總數(shù)及中性細(xì)胞比值均較緩解期明顯升高（P0.05）；經(jīng)簡(jiǎn)單相關(guān)分析可知：急性期和緩解期的IL-8均與中性粒細(xì)胞比值成正相關(guān)（急性期，r=0.463，p=0.046；緩解期，r=0.547，p=0.015）；急性期和緩解期的中性粒細(xì)胞數(shù)與TNF-α的含量呈正相關(guān)（急性期r=0.489，P=0.040；緩解期r=0.794，P=0.000）。通過(guò)多元逐步回歸分析發(fā)現(xiàn)，痰液MUC1水平受痰白細(xì)胞總數(shù)、FVC和年齡的影響。 二、熏煙動(dòng)物模型實(shí)驗(yàn)結(jié)果 1、用單純熏煙成功建立了COPD小鼠模型，其肺功能檢測(cè)顯示：氣道阻力顯著增加，肺順應(yīng)性、吸氣峰流速和呼氣峰流速顯著下降，結(jié)果較正常組小鼠相比，差異具有統(tǒng)計(jì)學(xué)意義；且光鏡下觀(guān)察COPD小鼠肺組織的病理改變可見(jiàn)：支氣管粘膜和肺實(shí)質(zhì)中均有大量炎癥細(xì)胞浸潤(rùn)，肺組織肺泡結(jié)構(gòu)大小不一，部分肺泡間隔斷裂，肺泡融合，肺泡腔擴(kuò)大，呈現(xiàn)出典型的肺氣腫病理改變。 2、 COPD小鼠肺組織及BALF中Muc1表達(dá)量明顯高于對(duì)照組正常小鼠，其差異具有統(tǒng)計(jì)學(xué)意義。COPD小鼠肺泡灌洗液中細(xì)胞總數(shù)、中性粒細(xì)胞數(shù)及巨噬細(xì)胞數(shù)均較對(duì)照組增加，TNF-α含量也較對(duì)照組明顯增加，差異均具有統(tǒng)計(jì)學(xué)意義（P0.05）。 三、急性炎癥動(dòng)物模型實(shí)驗(yàn)結(jié)果 1、運(yùn)用脂多糖滴鼻成功建立急性炎癥小鼠模型，光鏡下觀(guān)察模型組小鼠肺組織病理切片可見(jiàn)：肺間質(zhì)明顯水腫，肺實(shí)質(zhì)和支氣管粘膜中可見(jiàn)大量炎性細(xì)胞浸潤(rùn)。 2、經(jīng)western blot檢測(cè)小鼠肺組織Muc1胞內(nèi)片段水平，脂多糖處理12h后小鼠肺Muc1胞內(nèi)片段水平明顯升高，且具有統(tǒng)計(jì)學(xué)意義；24h后已下降至與對(duì)照組無(wú)統(tǒng)計(jì)學(xué)差異，脂多糖處理48h、96h和1w后的小鼠肺中已檢測(cè)不到Muc1胞內(nèi)片段變化（與對(duì)照組相比）。BALF中Muc1胞內(nèi)片段的表達(dá)量在各時(shí)間段一直處于較低水平，與對(duì)照組相比，無(wú)明顯變化。然而，BALF中Muc1的胞外片段水平在12h時(shí)最高，24h開(kāi)始下降，1周后下降至正常水平。脂多糖處理12h，24h，48h，96h，1w后的小鼠肺泡灌洗液中的細(xì)胞總數(shù)、中性粒細(xì)胞百分比均明顯升高，較對(duì)照組相比差異具有統(tǒng)計(jì)學(xué)意義（P0.01）。并且，各時(shí)間段的肺泡灌洗液中TNF-α的含量均較對(duì)照組升高。 四、細(xì)胞實(shí)驗(yàn)結(jié)果 分別用香煙煙霧提取物和脂多糖直接刺激A549細(xì)胞，24小時(shí)后收集細(xì)胞蛋白，均發(fā)現(xiàn)其中MUC1的表達(dá)量升高。 【實(shí)驗(yàn)結(jié)論】： 1、COPD患者氣道粘膜上皮MUC1的表達(dá)量升高。 2、COPD患者急性期痰液的MUC1表達(dá)較緩解期痰液高。 3、MUC1水平升高與脂多糖和香煙煙霧提取物直接刺激有關(guān)。 4、COPD患者的MUC1水平的升高可能與長(zhǎng)期煙霧刺激導(dǎo)致氣道慢性炎癥或細(xì)菌等刺激導(dǎo)致急性炎性相關(guān)。炎癥反應(yīng)誘導(dǎo)的的炎癥介質(zhì)升高，如TNF-α，以及中性粒細(xì)胞產(chǎn)物的增加可能與MUC1水平上調(diào)相關(guān)。
[Abstract]:Background Study Background :

Chronic obstructive pulmonary disease ( COPD ) is one of the chronic diseases with high morbidity and mortality in the world . It is characterized by progressive irreversible chronic airflow and is related to abnormal inflammatory response caused by smoking or inhalation of harmful particles or gases .

Mucin MUC1 ( human MUC1 indicates that animal Muc1 ) is an I - type transmembrane glycoprotein , which is divided into extracellular region , transmembrane region and intracellular region . It is mainly distributed in respiratory tract , gastrointestinal tract , reproductive tract epithelial cell and immune - related cell surface .

However , in COPD patients with chronic pulmonary inflammation , the level of MUC1 expression in the lung , whether expressed or not related to the level of inflammatory factors , is not yet clear .

Purpose of this study :

In this study , we studied the expression of MUC1 in lung and bronchial alveolar lavage fluid induced by chronic obstructive pulmonary disease ( COPD ) , airway mucosa and lipopolysaccharide - induced acute inflammatory animal lung tissue and bronchoalveolar lavage fluid in COPD patients .

Methodological Study Methodology :

The expression level of TNF - 偽 and IL - 8 in sputum was measured by means of Western blot and ELISA . ( 2 ) The expression level of TNF - 偽 and IL - 8 in sputum was detected by Western blot and ELISA .
Meanwhile , the levels of TNF - 偽 in the alveolar lavage fluid of mice were detected .
( 4 ) Using the method of cell culture , the cell protein was collected after 24 hours after stimulation of A549 cells with cigarette smoke extract and lipopolysaccharide , and the expression level of MUC1 was detected by Western blot .

The results of the experiment are as follows :

I . Increased expression of MUC1 in the airway mucosa of patients with COPD

1 . The expression of MUC1 expression in the airway mucosa of COPD patients was positive and the expression of normal airway mucosa was increased .

2 . During the acute stage of COPD , the expression of MUC1 in the supernatant of the supernatant of MUC1 was higher than that in the remission stage , and the levels of IL - 8 and TNF - 偽 in sputum were higher than those in the remission stage ( P0.05 ) .
The total number of white blood cells , the total number of neutrophils and the ratio of neutrophils in the acute period were significantly higher than those in the remission stage ( P0.05 ) .
The simple correlation analysis showed that IL - 8 in acute phase and remission stage was positively correlated with neutrophil percentage ( acute phase , r = 0.463 , p = 0.046 ) .
Response period , r = 0.547 , p = 0.015 ) ;
The neutrophil count in acute phase and remission stage was positively correlated with the content of TNF - 偽 ( acute stage r = 0.489 , P = 0 . 040 ;
Response period r = 0.794 , P = 0.000 ) . Multiple stepwise regression analysis found that the level of MUC1 in sputum was affected by the total number of sputum leukocytes , FVC and age .

II . Experimental results of smoking animal model

1 . The model of COPD mice was successfully established by simple smoking . The pulmonary function test showed that the airway resistance was significantly increased , the lung compliance , the inspiratory peak flow rate and the expiratory peak flow rate decreased significantly , and the difference was statistically significant compared with the normal group mice .
The pathological changes of lung tissues of COPD mice were observed under light microscope . There were a large number of inflammatory cell infiltration in the bronchial mucosa and the lung parenchyma , the alveolar structure of the lung tissue was different , some of the alveolar spaces were broken , the alveolar fusion and the alveolar cavity were enlarged , showing a typical pathological change of emphysema .

2 . The expression of Muc1 in the lung tissues and BALF of COPD mice was significantly higher than that in the control group . The total number of cells , the number of neutrophils and the number of macrophages in the alveolar lavage fluid of COPD mice were significantly higher than those in the control group , and the TNF - 偽 content was significantly higher than that of the control group ( P0.05 ) .

III . Experimental results of animal model of acute inflammation

1 . The acute inflammatory mouse model was successfully established by using lipopolysaccharide nasal drops . The pathological sections of lung tissues in the model group were observed under the light microscope . The pulmonary interstitial edema , pulmonary parenchyma and the infiltration of inflammatory cells were seen in the bronchial mucosa .

2 . The level of intracellular fragment of Muc1 in lung tissue was detected by western blot . After 12 hours of LPS treatment , the level of intracellular fragment of Muc1 in lung tissue was significantly increased , and it was statistically significant .
The expression of Muc1 in BALF was lower than that in the control group after 24h , 96h and 1w . The total number of cells and neutrophils in BALF were significantly higher than those in the control group ( P0.01 ) , and the levels of TNF - 偽 in the alveolar lavage fluid of each time period were higher than those in the control group .

IV . Cell experimental results

A549 cells were stimulated with cigarette smoke extract and lipopolysaccharide respectively . After 24 hours , the expression of MUC1 was increased .

Conclusion :

1 . The expression of MUC1 was increased in patients with COPD .

2 . The MUC1 expression of sputum in the acute stage of COPD patients was higher than that in the remission stage .

3 . The increased level of MUC1 was associated with direct stimulation of lipopolysaccharide and cigarette smoke extract .

4 . Increased levels of MUC1 in patients with COPD may be associated with chronic inflammation of the airways or irritation of bacteria , etc . due to long - term smoke stimulation . Inflammatory mediators - induced inflammatory mediators such as TNF - 偽 , and increase in neutrophils may be associated with up - regulation of MUC1 .
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R563.9

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