小鼠胃液誘導免疫細胞調節發炎的發生

Pro-inflammatory effect of gastric fluid on Raw264.7 cells

林漳聲(Chang-Sheng Lin)

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

氣喘是一種慢性呼吸道發炎反應，會造成呼吸道阻塞、過度反應和長期發炎等症狀。當呼吸道受到過敏原的刺激後產生過敏反應，呼吸道平滑肌會收縮並且大量分泌黏液，血液中的嗜中性白血球、嗜酸性白血球、淋巴球及單核球等免疫細胞會趨化且浸潤至呼吸道粘膜下。最後造成氣喘病人呼吸道狹窄阻塞及敏感度大增，粘膜水腫。胃食道逆流是指當胃液(包含胃酸及胃蛋白酶)不正常逆流至食道、咽喉，甚至進入氣管，會導致各部位不同程度的發炎，而產生逆流性食道炎的症狀。在過去的研究中指出，少量的胃液流入氣管中，可能破壞黏膜層，刺激組織產生發炎反應，但對於其中調節的機制仍尚未清楚。
首先，本實驗以胃液刺激小鼠巨噬細胞，發現可以成功的誘導老鼠腫瘤壞死因子(TNF-α)的表現，進而測試其他發炎因子也觀察到前列腺素E2 (PGE2)與一氧化氮(NO)的產生，證實了胃液的處理確實可以引起免疫細胞產生發炎的現象。甚之，以胃液刺激人類單核球細胞也發現，雖然無法造成其分化的現象，卻可以刺激細胞的增生。綜合以上，胃液可有效的誘導免疫球細胞調節發炎反應的產生。繼之，胃液的刺激也可以活化NF-?羠調控免疫反應的訊息傳導。有趣的是，在免疫反應發生時，通常發炎組織可以產生許多細胞激素，趨化免疫細胞移動到發炎的區域，因此我們以胃液刺激小鼠細胞，結果也顯示可以增加細胞移動的能力，再者，我們以胃液刺激大鼠氣管平滑肌細胞，接著共同培養小鼠巨噬細胞，也發現胃液的處理可以使平滑肌細胞產生許多調控因子，誘導小鼠細胞的移動。在過去的文獻中指出，細胞的轉移與胞外基質水解蛋白酶 (matrix metalloproteinase，MMP)有很大的關連性，我們以明膠蛋白酵素電泳分析法 (Gelatin zymography)，來測試經由小鼠胃液處理後，MMP蛋白的活性，結果顯示胃液可以刺激小鼠巨噬細胞表現MMP-9蛋白。更進一步，我們建構MMP-9 promoter–pGL3-basic 報告質體，也發現胃液可以調控MMP-9啟動子，增加冷光蛋白的表現量，當我們將啟動子上NF-?羠結合區域剔除後，也發現胃液的處理與控制組之間並無顯著差異，證實小鼠胃液可以透過NF-?羠的訊息傳導路徑來調控MMP-9的表現。因此，本實驗推論胃食道逆流症發生時，少量的胃液流入氣管中，確實可以刺激呼吸道的免疫細胞產生發炎的現象，進而增加氣喘發生的機率，同時也提供了一個新的方向，來治療由胃食道逆流症(gastroesophageal reflux disease, GERD)所引起的氣喘疾病。

關鍵字

胃食道逆流症 ； 老鼠腫瘤壞死因子

並列摘要

Asthma is known as a multiple factors, chronic inflammation airway disease. It is a complex and characterized by variable and recurring symptoms, airflow obstruction, bronchial hyper- responsiveness, and an underlying inflammation. Implications of micro aspiration of gastric fluid by GERD (Gastroesophageal Reflux Disease) and asthma has been reported in the past decades. However, little scientific evidence and mechanism are known for gastric-fluid-stimulated immune cells involved in bronchial disease. Therefore, the purpose of this study is to investigate the possible inflammatory effect of gastric fluid in murine macrophage cells and human monocytes. In the present study, we had set up an in vitro inflammatory model for the study of gastric fluid. Pro inflammatory stimulation of Raw264.7 cells was activated by gastric fluid collected from Balb/c mouse. We had evaluated the biological effect of gastric fluid in pro inflammatory cytokine TNF-alpha expression, PGE2 expression, cell proliferation, cell migration, MMP-9 expression, and NF-?羠 signaling pathway in Raw264.7 cells and THP-1 cells. In cells stimulated with gastric fluid, the nuclear translocation of NF-?羠 signaling component, p65, was increased. The gastric fluid can stimulate the cell proliferation and migration. The MMP-9 enzyme activity was enhanced by the treatment of gastric fluid, and the MMP-9 transcription regulation in association with the NF-?羠 signaling transduction activation was induced in the presence of gastric fluid. Taken together, the signaling components in NF-?羠 pathway or regulation of MMP-9 enzyme activity are potential targets for anti inflammation therapy of GERD.

並列關鍵字

GERD ； MMP-9

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