臺灣博碩士論文加值系統

背景與目標：越來越多證據顯示膽酸吸入肺部與許多肺部疾病具相關性，然而肺泡上皮細胞在接觸膽酸之後的反應卻仍然未知，因此我們假設膽酸對肺泡上皮細胞之刺激可能增加上皮細胞通透性，而導致肺傷害之形成。

方法：人類肺泡上皮細胞可在培養皿上生長成單細胞層，之後加入膽酸主成分之一的鵝去氧膽酸（chenodeoxycholic acid，CDCA）刺激，並觀察其上皮通透性之變化，而通透性之改變則可藉由經上皮層電阻（transepithelial electrical resistance，TER）之高低與小分子物質流經上皮細胞周圍間隙之能力－細胞旁通量（paracellular flux）來評估。在上皮細胞以CDCA刺激後，我們測量前列腺素E2 (prostaglandin E2，PGE2) 之產生，並評估以CDCA與PGE2刺激肺泡上皮細胞後，其通透性與細胞接合蛋白（junctional proteins）之變化。此外，我們亦藉由反轉錄聚合酶鏈式反應（reverse transcription-PCR）與西方墨點法（western blots）來顯示信息核醣核酸（mRNA）與接合蛋白之表現量。

結果：CDCA可顯著誘導p38與JNK蛋白之磷酸化反應，胞漿型磷脂水解酶A2 （cytosolic phospholipases A2, cPLA2）與環氧合酵素-2（cyclooxygenase-2, COX-2）之mRNA表現，PGE2之產生，TER之下降，與接合蛋白之衰減（接合蛋白包括occludin，zonula occludens-1 [ZO-1]，與E-cadherin，其中，ZO-1所產生之變化最顯著）。CDCA之刺激可造成細胞旁通量之增加，而此變化可因dexamethasone之前處理而減少。CDCA與PGE2兩者皆可造成TER之下降，而兩者下降之趨勢一致且呈現劑量-反應（dose-response）之相關變化。此外，PGE2與CDCA皆可造成ZO-1衰減，且兩者變化之趨勢非常相近。上皮細胞若以dexamethasone或特定蛋白抑制劑包括SB203580 (抑制p38)、SP600125 (抑制JNK)、mepacrine (抑制cPLA2)與NS398 (抑制COX-2)前處理，則可逆轉CDCA刺激所導致之PGE2生成、TER降低與ZO-1衰減。

結論：肺泡上皮細胞通透性之增加與接合蛋白之衰減有關。膽酸可經由調控MAP激酶、cPLA2、COX-2、PGE2及接合蛋白而改變上皮細胞之通透性，這些變化可能為膽酸導致肺傷害之機制。

Background and objective: Increasing evidence has shown that bile acid (BA) aspiration is associated with various lung diseases. The reaction of alveolar epithelium exposed to BAs is unknown. We hypothesize that BAs may induce alveolar permeability alteration and contribute to the pathogenesis of lung injury.

Methods: Human alveolar epithelial cells were grown in monolayer and stimulated with a major component of BAs, chenodeoxycholic acid (CDCA). Transepithelial electrical resistance (TER) and paracellular fluxes were measured to assess permeability alteration. PGE2 production was measured, and its effect on TER and junctional proteins (JPs) was also examined. Reverse transcription-PCR and western blots were used to investigate the expression of mRNA and JPs.

Results: CDCA induced significant p38 and JNK phosphorylation, cPLA2 and COX-2 mRNA expression, PGE2 production, TER reduction, and decay of JPs (including occludin, zonula occludens-1 [ZO-1], and E-cadherin, in which ZO-1 had maximal change). CDCA also increased paracellular fluxes, which was abolished by dexamethasone. Both CDCA and PGE2 contributed to TER reduction in an identical trend and a dose-response manner. PGE2 also reduced ZO-1 expression, which was similar to that observed by CDCA stimulation. Pretreatment with inhibitors of p38
(SB203580), JNK (SP600125), cPLA2 (mepacrine) and COX-2 (NS398) as well as dexamethasone reversed the CDCA-induced PGE2 production, TER reduction and decay of ZO-1.

Conclusions: The increase in alveolar permeability was associated with decay of JPs.BAs may induce permeability alteration through the upregulation of MAPK, cPLA2, COX-2, PGE2 and JPs, which may contribute to the pathogenesis of BA-associated lung injury.

一、英文縮寫……………………………………………………………........1
二、中文摘要……………………………………………………………………….3
三、英文摘要……………………………………………………………………….6
四、緒論….…………………………………………………………………………9
五、方法……………………………………………………………………….. 12
六、結果………………………………………………………………...………20
七、討論………………………………………………………………...………25
八、參考文獻………………………………………………………….………..29
九、圖表………………………………………………………………...………35
十、補充資料…………………………………………………………...………50
十一、英文論文…………………………………………………...……....56

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