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研究生:唐健豪
研究生(外文):tang chien hao
論文名稱:探討人類凝血酶調節素調控凝血酶訊號傳遞的作用機制
論文名稱(外文):Study on the modulating mechanism of human thrombomodulin on thrombin activated signaling
指導教授:張清堯黃蕙君黃蕙君引用關係
指導教授(外文):chang ching yaohuang hui chün
學位類別:碩士
校院名稱:亞洲大學
系所名稱:生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:80
中文關鍵詞:凝血酶蛋白酶活化接受器凝血酶調節素
外文關鍵詞:Thrombinprotease activated receptorsThrombomodulin
相關次數:
  • 被引用被引用:0
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  • 下載下載:49
  • 收藏至我的研究室書目清單書目收藏:1
凝血酶(Thrombin)是促進血液凝固的蛋白質,另外具有刺激血小板聚集、刺激細胞生長、活化免疫反應等的活性。凝血酶調控細胞生物活性,是分別透過結合蛋白酶活化接受器(protease activated receptors ; PARs)及凝血酶調節素(Thrombomodulin ; TM)二種不同接受器。本論文的主要目的在探討PAR 與TM 傳遞凝血酶訊號之機制差異。我們利用表現PAR-1 的人類胚胎腎臟細胞(human embryonic kidney ; HEK293 cells)轉染TM 基因為研究模式,轉殖TM 基因後之 HEK293 細胞命名為HEK293TM,觀察細胞型態及生長,發現HEK293TM 細胞型態聚集,生長速率約為HEK293 細胞之1/2。在凝血酶誘導的細胞爬行分析中,HEK293TM 細胞爬行能力較HEK293 增高為1.5 倍,ERK(Extracellular regulated kinase) 抑制劑U0126 , 可抑制HEK293TM 及HEK293 細胞爬行。分析TM 表現對凝血酶相關訊號機制之影響,HEK293 細胞在7.5 nM 凝血酶刺激20 分鐘後活化ERK,磷酸化提高為刺激前之1.5 倍,60、120、240 分鐘後之ERK 磷酸化逐步降低至基本值,而HEK293TM 細胞中ERK 有3 倍的活化,且磷酸化持續到4小時;同樣於HEK293TM 細胞,凝血酶激活PKB/Akt(protein kinase B)或PKC(protein kinase C)的能力也較HEK293 細胞高,且持續至4小時。進一步探討PKC 與ERK 間訊號路徑之交互作用,使用PKC 活性抑制劑Ro318220,發現並未影響HEK293TM 細胞中ERK 之活化及延長磷酸化,顯示PKC 未參與TM 所造成的ERK 延長磷酸化。TM 表現可以提升凝血酶經由PAR-1 傳遞的ERK、PKC 等活化訊號,造成細胞聚集,抑制細胞的生長,此外,PAR-1 及TM 經由活化ERK 而媒介凝血酶之促使細胞爬行活性。
Thrombin, a multifunctional serine protease generated at sites of vascular injury, induces multiple phenotypic changes of blood and vascular cells to affect vascular tone, cell permeability and growth, and leukocyte trafficking. Thrombin mediates cellular events by signal transduction through two different types of thrombin receptors: protease-activated receptors (PARs) and thrombomodulin (TM). Comparison of thrombin-PAR1 as to their ability to induce proliferation, thrombin-TM complex seems to restrains cell proliferation. These regulatory processes may rely on cross-talks between TM and PARs. We investigated the TM-modulating role on
thrombin-induced signal transduction pathways in PAR1-expressed human embryonic kidney (HEK293) cells transfected with TM. In the present works, TM-expressing cells (HEK293TM) cells grew in culture as close clustered colonies, while HEK293 cells dispersed in culture. TM expression resulted in a significant decrease of cell proliferation of HEK293 cells.In vitro analysis of cell migration by wound assay showed 1.5 fold accelerated migration of the HEK293TM compared to HEK293 cells. The activation of signaling was analyzed by Western blotting using an antibody specific for the phosphorylated forms of extracellular signal–regulated kinases (ERKs), protein kinase C (PKC), and protein kinase B (PKB), key signaling events central to the action of thrombin have been identified. 7.5 nM thrombin induced transient phosphorylation of ERKs in HEK293 cells with a peak at 20 min, whereas HEK293TM cells enhanced and sustained the phosphorylation of ERKs. Increased level of ERKs phosphorylation remained on activation state at 4 hours after thrombin stimulation. Thrombin also induced a higher phosphorylation of PKC or PKB/Akt in HEK293TM compared with control HEK293 cells. Add PKC inhibitor (3 μM Ro318220) after thrombin did not alter ERK phosphrylation prolonged .The results suggested that TM could modulate signaling through PAR-1 and the regulatory function of TM might in involved in sustained ERKs phosphorylation.
目錄 1
致謝 2
中文摘要 3
英文摘要 5
縮寫檢索表 8
一、背景 10
1.1 凝血酶 10
1.2 人類凝血酶調節素 12
1.3 蛋白酶活化接受器 15
1.4 細胞內之訊號調控分子 17
二、研究目的 21
三、研究材料 22
3.1 實驗儀器 22
3.2 實驗材料 24
四、實驗方法 28
4.1 細胞培養 28
4.2 轉染HEK293 細胞 31
4.3 蛋白質收集 32
4.4 蛋白定量濃度 34
4.5 西方墨點法分析 34
4.6 細胞型態 42
4.7 細胞活性測試 43
4.8 細胞爬行分析 44
五、實驗結果 45
5.1 篩選穩定表現TM 基因之轉殖細胞株 45
5.2 型態觀察與生長變化 45
5.3 觀察細胞爬行分析 46
5.4 凝血酶誘導HEK293TM 與HEK293 細胞訊號活化情形 46
六、討論 49
七、參考文獻 51
八、圖表 59
九、附錄 73
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