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研究生:賴璟暉
研究生(外文):Jing-Huei Lai
論文名稱:Nodal在人類惡性神經膠質瘤細胞調控細胞侵襲與增生所扮演的角色
論文名稱(外文):Role of Nodal in regulating cell invasiveness and growth in human glioma cells
指導教授:李宏謨李宏謨引用關係
指導教授(外文):Horng-Mo Lee
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學科學研究所
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:2008
畢業學年度:96
語文別:中文
論文頁數:55
中文關鍵詞:人類惡性神經膠質瘤細胞
外文關鍵詞:human glioma cells
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人類惡性神經膠質瘤細胞的特性是不受調控的快速增生和強烈的侵襲能力。本研究証實在不同的人類惡性神經膠質瘤細胞株有不同的增生速率和侵襲能力,而Nodal的蛋白質和mRNA表現跟細胞的增生及侵襲有正相關性。我們發現給予Nodal重組蛋白會造成的細胞的MMP-2活性和侵襲能力增加,顯示Nodal可能會調控細胞的MMP-2活性和侵襲能力。對人類惡性神經膠質瘤使用siRNA減低Nodal的表現則能有效的降低MMP-2活性和減低細胞的侵襲能力,並可以減緩腫瘤的增生與延長老鼠的存活率。這些結果顯示Nodal在人類惡性神經膠質瘤細胞增和和侵襲扮演重要角色。在使用MAPK的抑制劑或過度表現MKP-1,均可減少Nodal的產生。相反的,轉殖dominant negative MKP-1則無此效應。綜合以上結果可以得知,Nodal的訊息路徑在人類惡性神經膠質瘤細胞進程中扮演非常重要角色,而MKP-1可以調控經由Nodal所誘發的人類惡性神經膠質瘤細胞侵襲及增生當做重要治療標靶。
Human malignant glioma cells are characterized by uncontrolled growth and rapid invasion of adjacent tissues. In the present studies, we demonstrated that different human glioma cells exhibit distinct capacity of invasiveness and proliferation in vitro and in vivo. In addition, Nodal protein and mRNA levels correlate well with their capacity of proliferation and invasiveness. Treatment of human glioma cells with recombinant Nodal protein, we found MMP-2 activity and cell invasiveness were significantly increased. These evidences suggest that Nodal may regulate MMP-2 activity and cell invasiveness. When glioma cells were stable expressed with specific shRNA to knockdown Nodal expression, subsequently MMP-2 activity and expression were dramatically reduced. Nodal knockdown also reducd cell invasiveness and proliferation in glioma cells. In animal model, knockdown of Nodal expression resulted in decreasing of tumor growth in vivo and prolong animal survival rate. These results suggest that Nodal may play a role in glioma cell invasiveness and proliferation. Nodal expression is regulated by MAPKs activity. Ectopic expression of MKP-1 decreased Nodal expression. Expression of dominant negative MKP-1 did not alter the expression of Nodal. These data suggest that MKP-1 might reduce Nodal expression. Taken together, Nodal signaling pathway may play an important role in glioma progression involved in cell invasiveness and tumor growth. Given Nodal expression can be regulated by MKP-1, or incubation with pharmacological inhibitors specific to MAPKs. MKP-1 may serve as an important therapeutic target in Nodal-induced malignant glioma.
英文摘要 I
中文摘要 II
目錄 III
圖目錄 V

壹、 緒論 1
一、惡性腦瘤 1
二、基質金屬蛋白脢 (MMP) 1
三、Nodal 3
四、Rosiglitazone 4
五、MAP kinase phosphatase-1 (MKP-1) 4

貳、 研究材料與方法 6
ㄧ、研究材料 6
(ㄧ)、實驗細胞株 6
(二)、藥品試劑 6
(三)、常用儀器 8
(四)、常用溶液 9
二、研究方法 12
(ㄧ)、神經膠質瘤細胞的培養(cell culture in human
gliomas) 12
(二)、細胞蛋白質製備 (preparation of cell lysate)12
(三)、蛋白質定量法 (protein assay) 12
(四)、蛋白質電泳 (western blotting) 13
(五)、細胞存活率分析方法 (MTT assay) 13
(六)、Gelatin Zymography 14
(七)、載體轉殖(Plasmid transfection) 14
(八)、細胞侵襲性實驗 (Invasion assay) 14
(九)、反轉錄放大反應(Reverse Transcriptase-
polymerase Chain Recation) 15
(十)、活體侵襲實驗(In vivo intravasation assay)16
(十一)、腫瘤異體移植(Tumor xenograft assay) 17
(十二)、組織化學染色 (Histochemistry stain) 17

参、實驗結果與分析 18
一、探討Nodal與臨床惡性腦瘤之相關性 18
二、探討Nodal與惡性腦瘤細胞增生速率與侵襲能力之相關性: 18
三、探討Nodal在惡性腦瘤侵襲能力的角色 19
四、探討減低Nodal的表現是否會降低在惡性腦瘤侵襲能力 20
五、探討Nodal的表現是否會影響在動物模式中腦瘤侵襲能力與動物
存活率 21
六、細胞調控Nodal的機制 21
肆、結論與討論 23

伍、參考文獻 27

實驗結果圖表 34
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