胃液對大鼠氣管平滑肌細胞發炎反應之探討

Pro-inflammatory Studies of Gastric Fluid in Rat Airway Smooth Muscle Cells

陳嘉雯(Chia-Wen Chen)

指導教授 ： 鄭智美

高雄醫學大學/生命科學院/生物醫學暨環境生物學研究所/碩士(2009年)

摘要

氣喘(asthma)是一種慢性呼吸道發炎疾病。當氣喘發生時，呼吸道的平滑肌細胞會過度收縮，使呼吸道變窄，空氣通過時發生阻礙進而產生呼吸困難的現象。反覆的慢性呼吸道發炎反應會惡化氣喘的病情，也會導致呼吸道平滑肌發生結構上的改變(airway remodeling)。許多研究指出，胃食道逆流症(gastroesophageal reflux disease)會惡化氣喘的症狀，可能會引起相關的發炎反應；因此，本研究的目的主要在探討，胃液在呼吸道發炎反應中所扮演的角色及對呼吸道結構改變的影響。由細胞激素分泌分析(cytokine array)的結果，發現胃液會刺激大鼠呼吸道平滑肌細胞分泌TNF-α、IL-1β、IL-4、IL-6等Th2細胞激素，以及Fractalkine (FKN)、CINC-3、LIX等細胞趨化素；而且胃液會抑制Th1細胞激素與TIMP-1的表現。接下來，欲探討胃液與呼吸道平滑肌結構改變的關係，使用細胞移動分析(migration assay)來分析細胞受到胃液刺激後的移動情形，胃液能有效的增加大鼠呼吸道平滑肌細胞的移動能力。此外，藉由明膠蛋白酵素電泳分析(gelatin gel zymography)，發現了胃液會促進大鼠呼吸道平滑肌細胞中matrix metalloproteinase-2(MMP-2)的活性。經由西方點墨法(western blot)偵測，也發現胃液會增加人類氣管上皮細胞以及大鼠呼吸道平滑肌細胞中protease-activated receptor-2(PAR-2)的表現，而PAR-2的活化被研究指出和呼吸道黏液分泌的訊息傳遞有關。先前研究指出，FKN參與在呼吸道發炎疾病中，與趨化肥大細胞釋放發炎物質有關；使用酵素連結免疫分析(ELISA)系統偵測呼吸道平滑肌細胞中FKN的表現，證實胃液刺激會使得大鼠呼吸道平滑肌細胞FKN蛋白質表現量上升；利用半定量PCR(semi-quantitative PCR)偵測大鼠呼吸道平滑肌細胞FKN mRNA的表現，實驗結果發現，胃液並沒有顯著的影響FKN mRNA的表現情形。在細胞移動分析實驗中發現，FKN會促進大鼠呼吸道平滑肌細胞的移動現象，濃度越高刺激效果也隨之增加，但仍沒有達到胃液的刺激效果。此外，FKN和TNF-α、IL-1β的協同作用也無顯著刺激大鼠呼吸道平滑肌細胞的細胞移動現象及MMP-2的活性，但在FKN和IL-1β的共同刺激下，會刺激matrix metalloproteinase-9(MMP-9)的活性。綜合以上的實驗結果，胃液的確參與在呼吸道平滑肌初期的發炎反應中，甚至有可能會影響氣喘病人呼吸道平滑肌細胞結構上的改變，進而惡化氣喘的病情。

關鍵字

氣喘 ； 胃食道逆流症 ； 胃液 ； 呼吸道平滑肌細胞 ； 呼吸道結構改變 ； 細胞移動 ； 基質金屬蛋白水解酶

並列摘要

Asthma is a chronic airway inflammatory disease. In asthma, airway smooth muscle contracts that could narrow the airway lumen and cause airflow obstruction and dyspnoea. Repeated airway inflammation may exacerbate the condition of asthma and also cause the structural changes of airway smooth muscle. Gastroesophageal reflux disease (GERD) may also exacerbate the condition of asthma and is indicated as the inflammatory factor in asthma by many investigators. Therefore, the propose of our study is to evaluate the pro-inflammatory role of gastric fluid in airway inflammation and airway remodeling. The cytokine array assay was used to assess the effect of gastric fluid in rat airway smooth muscle cells, and production of Th2-cytokines and chemokines was enhanced by gastric fluid. Cytokines, including, TNF-α, IL-1β, IL-4, and IL-6, were increased after gastric fluid treatment, and chemokines, such as Fractalkine (FKN)、CINC-3 and LIX were also enhanced. Furthermore, Th1 cytokines and TIMP-1 were inhibited by gastric fluid. We want to know the relationship between gastric fluid and airway remodeling. The migration assay was used to analyze the chemotaxis of rat airway smooth muscle cells. The chemotaxis of rat airway smooth muscle cells was also enhanced by gastric fluid. The activity of matrix metalloproteinase-2 (MMP-2) was induced upon stimulation of gastric fluid assessed by gelatin gel zymography. We also found that the expression of protease-activated receptor-2 (PAR-2) was increased by stimulation of gastric fluid. PAR-2 has been indicated to be involved in airway inflammation and bronchial hyperresponsiveness. In the previous report, FKN was indicated to be involved in airway inflammatory disease. The expression of FKN was confirmed by ELISA assay. The mRNA of FKN wasn’t induced by gastric fluid as detected by semi-quantitative RT PCR. In migration assay, FKN could enhance the chemotaxis of rat airway smooth muscle cells in a concentration dependent manner. However, the chemotaxis effect of FKN is weaker than gastric fluid. The synergistic of FKN、TNF-α and IL-1β didn’t significantly induce the chemotaxis of rat airway smooth muscle cells and the activity of MMP-2, but the activity of MMP-9 could be enhanced. Our data show that gastric fluid is involved in the initiation of inflammatory process of rat airway smooth muscle cells and is probably contributing to the pathophysiology of airway remodeling in asthma.

並列關鍵字

asthma ； Gastroesophageal reflux diseas ； gastric fluid ； airway smooth muscle cell ； airway remodeling ； cell migration ； matrix metalloproteinase

參考文獻

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