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研究生:洪偉珊
研究生(外文):Wei-Shen Hung
論文名稱:Disabled-2在腦下垂體似濾泡星狀細胞中之表現與角色探討
論文名稱(外文):Disabled-2 expression and function in pituitary folliculostellate TtT/GF cell line
指導教授:陳賽君陳賽君引用關係
指導教授(外文):Sai-KoongTan
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:61
中文關鍵詞:腦下垂體內吞作用脂多醣濾泡星狀細胞Dab2
外文關鍵詞:Dab2pituitaryLPSTtT/GFendocytosis
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腦下垂體是人體的內分泌中樞,發生發炎會引起腦下垂體內分泌失調,進而影響生理以及免疫系統的正常運作。腦下垂體似濾泡星狀細胞能調控隣近腦下垂體內分泌細胞之功能,因此研究腦下垂體似濾泡星狀細胞之發炎機制將有助於預防以及治療腦下垂體之發炎。Disabled-2是一種轉接蛋白,參與細胞內吞作用及訊號傳遞。我們發現LPS對腦下垂體似濾泡星狀細胞株TtT/GF所誘發的細胞激素基因表現會被細胞內吞作用抑制劑cytochalasin D所抑制,顯示LPS誘發細胞激素基因表現之訊息傳遞與內吞作用有關。研究更進一步發現Dab2基因於TtT/GF細胞中有表現,且Dab2 mRNA在LPS作用三小時後會明顯的降低,而細胞質內蛋白質表現量反而是明顯的增加。免疫螢光染色的結果觀察到Dab2蛋白以顆粒狀分佈於細胞質中,隨著LPS作用3至6小時,Dab2蛋白顆粒變粗,高密度聚集於核周圍Golgi區域,於12小時後此聚集現象即消失,又恢復原狀。以共軛焦螢光顯微鏡的結果確認Dab2蛋白高度聚集於核周圍,並不會進入細胞核內。利用帶紅色螢光之LPS分子以及外送綠色螢光之Dab2蛋白更進一步確認Dab2與LPS於細胞內座落於相同之位置。另外Dab2 siRNA 抑制Dab2蛋白表現,也阻斷了LPS所引發的細胞激素基因表現活化及細胞移動的行為。以上結果皆顯示Dab2參與LPS刺激腦下垂體似濾泡星狀細胞發炎之反應,可能在LPS被細胞內吞後活化發炎前基因表現以及細胞移動行為之訊號傳遞中扮演重要的角色。然而此現象在生理上的意義,尚需進一步的探討。
Disabled-2 (Dab2), an adaptor protein, is involved in various cell processes, including endocytosis. Here we found the blockade activationof lipopolysaccharide (LPS) -induced proinflammatory cytokine expression after cytochalasin D (endocytosis inhibitor) treatment which suggested LPS signaling is endocytic dependent in pituitary
folliculostellate (FS) cell line- TtT/GF. We further showed that Dab2 is expressed in TtT/GF cells. Dab2 mRNA level slightly decreased, but the protein level significantly increased after 3 hours LPS administration. Immunofluorescence analysis showed that Dab2 protein was found to be expressed intracellularly and formed enlarged clusters within perinuclear after 6 hours LPS treatment, following which it was no longer detectable in this region, but remaining fine granules in cytosol. Confocal immunofluorescence analysis showed that Dab2 was not translocated into the nuclear region. The granule-like distribution was further confirmed by over-expressing GFP-Dab2 into TtT/GF cells. Over-expressed GFP-Dab2 was co-localized to Alexa Fluor 594-LPS in TtT/GF cells. Transfected siDab2 cells blocked the LPS-induced pro-inflammatory cytokine expression and migration ability of TtT/GF cells. Although Dab2 did not co-localized with vesicle-transporter protein α-tubulin after LPS stimulation, these results suggest that Dab2 is important for the endocytic pathways of LPS signaling in pituitary folliculo-stellate cells.
中文摘要‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥3
英文摘要‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥4
緒論‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥5
研究目的‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥11
實驗方法‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥12
實驗結果‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥23
討論‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥29
結果圖表‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥33
附錄‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥47
參考文獻‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥55
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