Celecoxib抑制腎小管細胞經壓力所導致之轉化生長因子b表現及細胞外間質之生成

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題名:	Celecoxib抑制腎小管細胞經壓力所導致之轉化生長因子b表現及細胞外間質之生成 Celecoxib Inhibits Pressure-Induced TGF-beta Expression and Extracellular Matrix Synthesis in Renal Tubular Cells
作者:	鄭仲益 Chung-Yi Cheng
貢獻者:	醫學科學研究所
關鍵詞:	阻塞性腎臟病轉化生長因子b 細胞外間質壓力 obstructive uropathy TGF-bExtracellular matrix celecoxib pressure
日期:	2008
上傳時間:	2009-08-28 11:23:18 (UTC+8)
摘要:	背景轉化生長因子b在單側輸尿管阻塞之動物模式對於調控細胞外間質之生成扮演著重要的角色，過去的動物研究曾經指出選擇性第二型環氧合酶抑制劑於單側輸尿管阻塞之動物模式保護效果，但是對於環氧合酶如何保護的機制並不是清楚的被探討；我們利用加壓於腎小管細胞的實驗模式來探討轉化生長因子b在細胞外間質之生成所影響的效應，以及探討選擇性第二型環氧合酶抑制劑如何來抑制轉化生長因子b之表現。方法根據Broadbelt NV等作者的研究我們對於細胞加壓的實驗器具有所修飾，腎小管細胞NRK52E培養於25mm2的培養瓶，細胞接受於不同時間點60mmHg及120mmHg之壓力(30 分鐘-24小時)，細胞培養液收集後作酶聯免疫吸附試驗，而溶解於緩衝液之細胞以西方墨點法分析；單側輸尿管阻塞動物實驗前以每日投予celecoxib 10mg/kg連續7日後，腎臟於第7日及第14日取出作免疫組織化學分析。結果腎小管細胞以60mmHg加壓後8小時轉化生長因子b與細胞只有加壓30分鐘比較有意義的增加(約50倍增加)，而以西方墨點法分析，纖維粘連蛋白也在細胞加壓後8小時有意義的上升；Celecoxib對於阻塞性腎病變之保護效果我們也有所探討，我們發現隨著celecoxib治療劑量的增加轉化生長因子b的表現量逐漸的減少，最大抑制轉化生長因子b的表現在投予celecoxib 50mM，同時以免疫印記法表現投予celecoxib後纖維粘連蛋白也在細胞加壓後8小時明顯的減少；最後在動物實驗以病理表現celecoxib有效的減少間質性腎臟病變，並以免疫組織化學方式分析轉化生長因子b的在投予celecoxib後減少表現。結論細胞外間質的表現增生發生在單側輸尿管阻塞之動物實驗模式進而造成間質性纖維化，以細胞加壓模式模擬輸尿管阻塞所產生之壓力可導致細胞外間質的表現增生，以第二型環氧合酶抑制劑抑制轉化生長因子b的表現可以減少細胞外間質的增生。 Background. Transforming growth factor-beta （TGF-b）plays an important role for regulation of extracellular matrix expression in the Unilateral Ureteral Obstruction （UUO）model. Previous animal studies had shown the protective effect of selective cyclooxygenase-2（COX-2）inhibitor in the UUO. However, the exact cellular mechanism of COX-2 inhibitor protective effect was not known. We explored the effect of TGF-b on expression of extracellular matrix in the pressurized renal tubular cells, NRK-52E and hence, fibroprotective effects of COX-2 inhibitor, celecoxib upon suppression of TGF-b expression. Methods. We modified the novel pressure apparatus applied to the culture renal tubular cells (RTC) originally designed by Broadbelt NV et al. Renal Tubular Cells, NRK52E were cultured in 25mm2 flasks. Cells were subjected to pressure of 60 mmHg over time (30 min – 24 h) and 120 mmHg over the similar time interval. Cell lysates and cultural medium were collected used for Western blots and ELISA respectively. Daily dose of Celecoxib were administered everyday for 7 days prior to UUO, and kidneys were harvested at day 7 and day 14. Immunohistochemistry was used to reveal TGF-b expression in renal tubulointerstitial system. Results. TGF-b had significantly elevated 8h after cells were applied with pressure 60mmHg ( 50-folds of increase compared to baseline ) and decreased thereafter. Western blot revealed significant elevated fibronectin protein 8h after introduction of pressure applied to RTC. The protective effect of celecoxib on obstructive uropathy was tested. Level of TGF-b had shown progressively decrease in a dose dependent trend after treated with Celecoxib, the maximal effect was shown at 50mM of Celecoxib. Western blot further confirmed the protective effect of Celecoxib in this pressure model of obstructive uropathy by showing dose dependent decrease in fibronectin expression after Celecoxib treatment. In vivo study, we demonstrated with decreased degree of renal tubulointerstitial damage followed Celecoxib treatment. The level of TGF-b was decreased followed Celecoxib treatment evident by immunohistochemistry. Conclusion. Pressure application of NRK-52E results in extracellular matrix synthesis, a process which may be activated in UUO and contributes to interstitial fibrosis. Inhibition of cyclooxygenase-2 may reduce extracellular matrix synthesis through a TGF-b-dependent process.
資料類型:	thesis
顯示於類別:	[醫學科學研究所] 博碩士論文

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