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研究生:洪羿筑
研究生(外文):I-Chu Hong
論文名稱:Dexamethasone促進敗血症鼠肝中rBAT表達時,RXR所扮演之角色探討
論文名稱(外文):Role of retinoid x receptor in upregulation of hepaticrBAT by dexamethasone during sepsis
指導教授:許勝光
指導教授(外文):Hseng-kuang Hsu
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:醫學研究所碩士班
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:45
中文關鍵詞:敗血症糖質類固醇rBATRXR
外文關鍵詞:sepsisdexamethasonerBATretinoid x receptor
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敗血症會造成肝內膽汁鬱積 ( intra-hepatic cholestasis ),膽酸的運送功能及膽汁流速 ( bile flow )會減少,導致膽酸堆積(bile acid accumulation)的情形,進而引起肝細胞結構與生理功能的破壞。膽酸需要與胺基酸結合後形成膽鹽才被運送及分泌至膽管。在我們先前的研究結果顯示,敗血症時形成膽鹽之主控酵素rBAT的表現呈有敗血症特異性減少,值得我們繼續做深入的探討。因文獻指出rBAT基因的IR-1序列上,當結合上轉錄因子Farnesoid X receptor (FXR)和Retinoid X receptor(RXR)形成heterodimers後,才轉譯成rBAT蛋白質,續而影響膽酸的分泌;另外,也有文獻指出FXR與膽酸結合並和RXR形成heterodimers後,會調節使膽酸的主要運送者Bile Salt Export Pump(BSEP)之形成。故本實驗擬以敗血症之動物模式及肝細胞(hepatocyte cell)體外試驗分別探討在敗血症過程中FXR/RXR對rBAT及BSEP表現的調控情形。本研究期望對敗血症時rBAT、BSEP表現的分子機制之探討能在敗血症的病程指標及治療方法有些貢獻。
本研究之實驗技術與方法,簡單說明如下:本研究以盲腸結紮穿孔術(cecal ligation and puncture;CLP) 引發大白鼠敗血症之動物模式,首先在活體內實驗,分實驗組和偽手術組,分別於不同時間點(3,6,9,18 Hours)取部分肝臟 (左上葉),(1)用西方點墨法分別觀察FXR、RXR,rBAT及BSEP蛋白質表現的變化,(2)用RT-PCR偵測rBAT,BSEP 之mRNA表現的變化,(3)用EMSA來了解FXR及RXR結合至IR-1序列的活性之變化情形,由以上結果探討分析在敗血症病理中rBAT,BSEP 之mRNA或蛋白質表現的變化受到FXR及RXR的調控情形。接著分離老鼠肝臟的肝細胞(hepatocyte cell)進行活體外試驗,用西方點墨法、及EMSA等方法來探討分析FXR/RXR、 rBAT、及BSEP間的關係是否與體內模式相同,以確立活體外肝細胞之實驗模式,並探討dexamethasone改善rBAT 蛋白質表現之作用。
觀察到的結果如下,由活體內實驗我們得知: (1)以CLP model引發老鼠產生敗血症後,FXR/RXR結合至IR-1的能力下降,而其下游基因BAT和BSEP在蛋白質和mRAN的表現也有下降的現象(2)RXR蛋白質的表現量在CLP 6小時後就明顯下降,而FXR蛋白質的表現量在CLP 9小時後才有減少的趨勢。由以上結果我們繼續進行活體外實驗,在CLP 6小時後,取出老鼠肝細胞,得知給予Dexamethasone後發現: (1)RXR蛋白質的表現量的確有明顯的增加,且FXR/RXR結合至IR-1的能力上升(2) 其下游基因BAT蛋白質的表現有上升的現象,但對BSEP卻沒有影響。
由以上研究之結果,可做出以下之推論: 在敗血症時BAT和BSEP會受到FXR/RXR的調控,且RXR在敗血症時對rBAT調控,具有其特異性的角色,同時以體外肝細胞進行Dexamethasone的投予應能改善敗血症時的肝臟損傷,且其作用機轉可能為經由RXR對rBAT調控。
Severe infection and sepsis may cause intrahepatic cholestasis and lead to hepatic dysfunction. Our previous results showed that the expression of bile acid CoA:amino acid N-acyltransferase (rBAT), the enzyme required for bile salt formation,was significantly decreased in the liver at the early stage of sepsis. Additionally,bile salt export pump(BSEP),a bile salt export pump, was also suppressed under septic insult.Both the transcriptions of rBAT and BSEP are regulated by the heterodimer of farnesoid X receptor(FXR) and retinoid X receptor (RXR) via IR-1 sequence.Therefore,the present study aimed to investigate the possible role of FXR/RXR upon the expressions of rBAT and BSEP.
Animal model of sepsis was induced by cecal ligation and puncture (CLP).To demonstrate wheather rBAT and BSEP protein and mRNA are modulated by FXR/RXR in CLP model.Liver tissues were sampled at 3、6、9 and 18 hours after CLP operation.Western blotting,RT-PCR and EMSA were performed to estimate protein content (FXR,RXR,BSEP,rBAT),mRNA expressions (BSEP,rBAT) and DNA binding activities (FXR/RXR binding to IR-1),respectively.Otherwise,hepatocytes were isolated from the septic rats at 6 hours after CLP to study the in vitro effect of dexamethasone,which enhances RXR expression,on the protein expressions of rBAT and BSEP.
The results of in vivo experiments showed that: (1) The DNA binding activity of FXR/RXR was decreased at 6 hrs after CLP.Both protein and mRNA expression of rBAT、BSEP were also decreased.(2) Protein expression of RXR was decreased at 6 hrs after CLP,while FXR protein content was decreased at 9 hrs after CLP.We also tested the effect of dexamethasone of hepatocytes from the septic rats at 6 hrs after CLP.The results showed that: (1) Protein expression of RXR was increased and the DNA binding activity of FXR/RXR was also increased.(2) The protein expression of rBAT was significantly enhanced by dexamethasone treatment while no effect of dexamethasone on BSEP protein expression was observed.
We conclude that (1) The rBAT and BSEP were modulated by FXR/RXR in CLP model.(2) The early suppression of RXR by septic insult may play an important role in hepatic dysfunction via regulating rBAT but not BSEP expression.(3) Dexamethasone may improve liver function in sepsis, via RXR modulation on rBAT.
I. 中文摘要
II. 英文摘要
Ш. 緒論
一.敗血症與肝臟及肝中膽酸及膽鹽的合成、代謝及運送的關係
二.BAT之背景介紹
三.BSEP之背景介紹
四.Farnesoid X receptor (FXR)之簡介
五.Retinoid X receptor (RXR) 之簡介
六.研究主旨
IV.材 料
一.化學試劑與溶液
二.實驗動物
V.實驗方法
一. 引發敗血症之動物模式 (Animal Model of sepsis)
二. 分離肝細胞
三. 蛋白質濃度之測定
四. 西方墨點法 (Western blot)
五. RT-PCR
六. 電泳移動偏移分析法(EMSA)
VI. 結果
一. 盲腸結扎穿刺手術後對實驗動物的影響
二. 敗血症時,肝臟中BSEP蛋白質和mRNA之表現情形
三. 敗血症時,肝臟中FXR/RXR結合至IR-1序列上之表現情形
四、敗血症時,肝臟中FXR和RXR蛋白質之表現情形
五、體外肝細胞分離後的情形
六、CLP手術6小時後,肝細胞中RXR蛋白質之表現情形
七、肝細胞分離後給予Dexamethasone,肝臟中FXR/RXR結合至IR-1序列上之表現情形
八、敗血症所分離出的肝細胞中給予Dexamethasone後,其BAT蛋白質和BSEP蛋白質之表現情形
VII. 圖表
VIII. 討論
IX. 參考文獻
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