MUC1在慢性阻塞性肺疾病发病过程中的表达改变和初步机制研究

发布时间：2018-06-20 18:56

本文选题：慢性阻塞性肺疾病 + 痰液　；参考：《广州医学院》2012年硕士论文

【摘要】：【研究背景】：慢性阻塞性肺疾病(Chronic Obstructive Pulmonary Disease, COPD)是世界范围内高发病率和高死亡率的慢性疾病之一，它以渐进性不完全可逆的慢性气流受限为特征，并与吸烟或吸入有害颗粒或气体引起的异常炎症反应有关。大量研究证明，气道慢性炎症是COPD发病的核心内容，其形成机制十分复杂，涉及多种炎性细胞，在肺的不同部位有肺泡巨噬细胞、T淋巴细胞和中性粒细胞的增加，激活的炎症细胞释放各种炎性介质如TNF-α，IL-8，从而损伤肺组织，促进COPD的发生和发展。粘蛋白MUC1（人类用MUC1表示，，动物用Muc1表示）是一种I型跨膜糖蛋白，分为细胞外区、跨膜区和胞内区，主要分布在呼吸道、胃肠道、生殖道上皮细胞以及免疫相关细胞表面。它对正常上皮起润滑和保护作用，同时它的胞内区还可以介导信号转导和细胞粘附功能。近期研究表明不论是铜绿假单胞菌感染还是呼吸道合胞体病毒感染引起的急性炎症中，都可以观察到Muc1的表达量在感染后大量增加，而且这种表达增加与TNF-α、中性粒细胞弹性蛋白酶等的表达密切相关。从而提示Muc1是在感染和炎症发生时起作用的重要内源性分子。但在出现肺部慢性炎症的COPD患者中，肺中MUC1的表达水平如何，其表达水平是否也与炎症因子水平相关，目前尚不清楚。【研究目的】：本实验研究了不同来源标本，包括慢性阻塞性肺疾病（COPD）患者气道粘膜和不同时期的痰液标本，香烟烟雾诱导的COPD模型小鼠和细菌脂多糖诱导的急性炎症动物肺组织和支气管肺泡灌洗液，以及利用香烟烟雾提取物和细菌脂多糖直接刺激的A549细胞中MUC1的表达改变情况。并分别检测了COPD患者不同时期痰液中IL-8和TNF-α水平及COPD小鼠和急性炎症小鼠支气管肺泡灌洗液中TNF-α的水平。旨在揭示长期烟雾刺激和急性细菌脂多糖刺激后小鼠肺内Muc1的表达变化与TNF-α变化相关性，进而探讨COPD患者中MUC1的表达变化及其可能的机制，为COPD的发病机制提供新的理论基础，并为COPD的预防和治疗提供新的作用靶点。【研究方法】：本研究采用临床病人标本、动物实验和细胞培养相结合的方法。（1）通过纤支镜活检留取COPD患者的气道粘膜，采用免疫组化染色的方法来观察MUC1的表达水平。分别收集COPD病人急性期和缓解期的痰液标本，采用酶联免疫吸附法（ELISA）检测痰液上清中MUC1的量表达，同时检测痰液中TNF-α和IL-8的水平。（2）利用单纯熏烟6个月建立COPD小鼠模型，采用Western blot印迹法和ELISA检测COPD模型小鼠肺内Muc1表达水平，并检测支气管肺泡灌洗液中TNF-α的含量及细胞分类计数。（3）采用脂多糖滴鼻致敏C57小鼠后建立急性炎症动物模型，分别收集12h、24h、48h、96h、1w后的肺组织和支气管肺泡灌洗液，并分别采用Western blot和ELISA的方法检测各时间段的肺内Muc1的表达水平；同时检测小鼠肺泡灌洗液中TNF-α的水平；（4）采用细胞培养的方法，分别用香烟烟雾提取物和脂多糖刺激A549细胞24h后，收集细胞蛋白，采用Western blot的方法检测MUC1的表达水平。【实验结果】：一、COPD患者气道粘膜上皮MUC1的表达增加 1、在COPD患者的气道粘膜上皮MUC1免疫组化表达阳性且较正常气道粘膜的表达量增多。 2、 COPD患者急性期痰液上清中MUC1胞内片段表达较缓解期明显升高，且痰液上清IL-8水平、TNF-α水平均较缓解期升高，差异具有统计学意义（P0.05）；急性期痰液中白细胞总数、中性粒细胞总数及中性细胞比值均较缓解期明显升高（P0.05）；经简单相关分析可知：急性期和缓解期的IL-8均与中性粒细胞比值成正相关（急性期，r=0.463，p=0.046；缓解期，r=0.547，p=0.015）；急性期和缓解期的中性粒细胞数与TNF-α的含量呈正相关（急性期r=0.489，P=0.040；缓解期r=0.794，P=0.000）。通过多元逐步回归分析发现，痰液MUC1水平受痰白细胞总数、FVC和年龄的影响。二、熏烟动物模型实验结果 1、用单纯熏烟成功建立了COPD小鼠模型，其肺功能检测显示：气道阻力显著增加，肺顺应性、吸气峰流速和呼气峰流速显著下降，结果较正常组小鼠相比，差异具有统计学意义；且光镜下观察COPD小鼠肺组织的病理改变可见：支气管粘膜和肺实质中均有大量炎症细胞浸润，肺组织肺泡结构大小不一，部分肺泡间隔断裂，肺泡融合，肺泡腔扩大，呈现出典型的肺气肿病理改变。 2、 COPD小鼠肺组织及BALF中Muc1表达量明显高于对照组正常小鼠，其差异具有统计学意义。COPD小鼠肺泡灌洗液中细胞总数、中性粒细胞数及巨噬细胞数均较对照组增加，TNF-α含量也较对照组明显增加，差异均具有统计学意义（P0.05）。三、急性炎症动物模型实验结果 1、运用脂多糖滴鼻成功建立急性炎症小鼠模型，光镜下观察模型组小鼠肺组织病理切片可见：肺间质明显水肿，肺实质和支气管粘膜中可见大量炎性细胞浸润。 2、经western blot检测小鼠肺组织Muc1胞内片段水平，脂多糖处理12h后小鼠肺Muc1胞内片段水平明显升高，且具有统计学意义；24h后已下降至与对照组无统计学差异，脂多糖处理48h、96h和1w后的小鼠肺中已检测不到Muc1胞内片段变化（与对照组相比）。BALF中Muc1胞内片段的表达量在各时间段一直处于较低水平，与对照组相比，无明显变化。然而，BALF中Muc1的胞外片段水平在12h时最高，24h开始下降，1周后下降至正常水平。脂多糖处理12h，24h，48h，96h，1w后的小鼠肺泡灌洗液中的细胞总数、中性粒细胞百分比均明显升高，较对照组相比差异具有统计学意义（P0.01）。并且，各时间段的肺泡灌洗液中TNF-α的含量均较对照组升高。四、细胞实验结果分别用香烟烟雾提取物和脂多糖直接刺激A549细胞，24小时后收集细胞蛋白，均发现其中MUC1的表达量升高。【实验结论】： 1、COPD患者气道粘膜上皮MUC1的表达量升高。 2、COPD患者急性期痰液的MUC1表达较缓解期痰液高。 3、MUC1水平升高与脂多糖和香烟烟雾提取物直接刺激有关。 4、COPD患者的MUC1水平的升高可能与长期烟雾刺激导致气道慢性炎症或细菌等刺激导致急性炎性相关。炎症反应诱导的的炎症介质升高，如TNF-α，以及中性粒细胞产物的增加可能与MUC1水平上调相关。
[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 - 伪 and IL - 8 in sputum was measured by means of Western blot and ELISA . ( 2 ) The expression level of TNF - 伪 and IL - 8 in sputum was detected by Western blot and ELISA .
Meanwhile , the levels of TNF - 伪 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 - 伪 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 - 伪 ( 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 - 伪 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 - 伪 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 - 伪 , and increase in neutrophils may be associated with up - regulation of MUC1 .
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563.9

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