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系統識別號 U0026-0812200911330444
論文名稱(中文) 探討 GCIP 與 ID蛋白質之間的交互作用及其生理影響
論文名稱(英文) Study on the Interaction between GCIP and ID Proteins, and It’s Biological Effect
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 93
學期 2
出版年 94
研究生(中文) 蔡宗霖
研究生(英文) Tsai-Tsung Lin
學號 S1692110
學位類別 碩士
語文別 中文
論文頁數 70頁
口試委員 口試委員-賴明德
口試委員-劉俊明
口試委員-張明熙
指導教授-張敏政
口試委員-張文粲
中文關鍵字 磷酸化  交互作用  細胞週期 
英文關鍵字 interaction  cell cycle  phosphorylation 
學科別分類
中文摘要 GCIP蛋白質其全名為human Grap2 and Cyclin D Interacting Protein。根據之前的研究,GCIP 蛋白質在人體的每個組織中都會表現;然而在一些分化到末期的組織中,例如心臟、肌肉、周邊血液系統中的白血球以及大腦,GCIP 蛋白質的表現會達到最高。除此之外,先前的研究發現,GCIP 蛋白質會減少pRB 蛋白質的磷酸化,並且抑制E2F1 轉錄因子的轉錄能力,而這兩個部分是廣泛被接受在調控細胞週期由 G1時期進行到S 時期中,最重要的調控步驟。
ID蛋白質(Inhibition of Differentiation and Inhibition of DNA binding)包含有四個成員分別為ID1、ID2、ID3及ID4。此類蛋白質在結構上具有一個HLH(Helix-Loop-Helix)區域但卻缺乏一個鹼性DNA鍵結(basic DNA binding)區域,也由於此特性使得ID蛋白質具有負調控鹼性轉錄因子(basic transcription factor)的功能。根據近幾年的文獻指出,ID蛋白質對於腫瘤生成的影響已經在許多人類的癌症疾病中被證實,因此,ID蛋白質已被視為是癌症治療及診斷工具的標的。
在本篇論文中,經由免疫沉澱法的結果驗證,GCIP蛋白質能與ID1、ID2及ID3蛋白質在細胞內(In vivo)與試管中(In vitro)的環境下產生交互作用。除此之外,利用Id1(Santa Cruz biotechnology)與實驗室製備的GCIP多株抗體於西方墨點法中發現,ID1蛋白質於非惡性(不具轉移性)的乳癌細胞(MCF-7)中可被血清所調控,但在惡性(具轉移性)乳癌細胞(MDA-MB 231)中卻不受血清的調控,此外,亦發現GCIP蛋白質只有在非惡性的乳癌細胞(MCF-7)中才有表現。
許多文獻中指出,ID2蛋白質具有使 retinoblastoma (pRb) 及調控抑制細胞週期蛋白質活性的能力,進而刺激細胞從G1期進入S期。在暫時轉染Id2 基因的細胞中,當 ID2 與 pRB 蛋白質之間形成鍵結時,會導致 pRB 對於抑制細胞的能力喪失,而在持續表現GCIP蛋白質的HEK-293T細胞中,卻發現此抑制現象被減弱。除此之外,在NIH-3T3的細胞中GCIP蛋白質亦可反轉E2F蛋白質從 pRB 被釋放的現象。
根據上面的實驗結果,我們認為GCIP蛋白質或許可分別經由調控ID1及ID2蛋白質所參與的訊息傳遞路徑來影響乳癌細胞的轉移及生長,除了ID1及ID2蛋白質外,對於其他ID蛋白質的成員仍需更進一步的研究探討。



英文摘要 GCIP is a novel human Grap2 and Cyclin D Interaction Protein, which was reported first in 2000. The expression of GCIP was found in all human tissues with the highest level of expression in heart, muscle, peripheral blood leukocytes and brain. Furthermore, overexpression of GCIP was found to reduce the phosphorylation of retinoblastoma (pRb) protein and inhibit E2F-1 mediated transcription activity, which is generally considered the critical step for cell cycle progression from the G1 phase into the S phase. The Id (inhibition of differentiation and inhibition of DNA binding) proteins have 4 members designated Id1- 4. They have a helix-loop-helix (HLH) domain but lock a basic DNA-binding domain, it makes them to act as a dominant negative antagonists of the basic helix-loop-helix family of transcription factors. Recently, The tumorigenesis effect of ID proteins has been demonstrated in a number of human cancers. Consequently, ID proteins are thought to be the targets for cancer therapy or diagnostic tool. In this study, immunoprecipitation experiments demonstrated that GCIP protein interacted with ID1、ID2 and ID3 proteins both In vivo and In vitro. In addition, by using Id1(Santa Cruz biotechnology) and GCIP (polyclonal) antibodies, we found that Id1 protein is serum dependent in less malignant phenotype breast cancer cell ( MCF-7,none metastasis cell ) but is serum independent in malignant phenotype breast cancer cell ( MDA-MB 231, metastasis cell ). Furthermore GCIP protein was detected in MCF-7 cell line but not in MDA-MB 231 cell line. It was reported that the Id2 protein has ability to inactive retinoblastoma (pRb) but also abrogate the cell cycle-inhibitory activity, and then stimulates the cell to pass the G1 phase into S phase. The interaction of Id2 with pRb resulted in a loss of pRb mediated growth suppression in HEK-293T cell which containing a transiently transfected Flag - myc tagged Id2, whereas GCIP protein reduced this suppression in HEK293T cell containing an overexpression of GCIP. In addition, the GCIP protein also could reverse the E2F to release from pRb in NIH3T3 cell. Taken together, this data suggested that GCIP might be involved in the regulation of metastasis in breast cancer cell and of cell’s proliferation through Id1 and Id2 mediated signaling pathway respectively. Besides the ID1 and ID2 proteins, the relationships between other Ids will also be investigated.


論文目次 中文摘要.......................................2
英文摘要.......................................4
目錄...........................................6
圖目錄.........................................7
縮寫檢索表.....................................9
緒論..........................................11
材料與方法....................................17
結果..........................................34
一、GCIP 蛋白對於細胞生長的影響.............34
二、GCIP 與Id蛋白在試管內(in vitro)和活體內(in vivo)交互作用的結果.........................35
三、偵測癌化程度不同的乳癌細胞株( MCF7 與MDA-MB 231)中內生性GCIP與Id1 蛋白的含量............37
四、Id2 蛋白對於細胞週期所產生的影響(負調控)可被GCIP 所反轉-對於細胞週期調控蛋白 Cyclin D1 的影響..........................................39
五、Id2 蛋白對於細胞週期所產生之影響(負調控)可經由 GCIP 而反轉-對於細胞週期調控蛋白Retinoblastoma (pRb) 的影響.................40
六、Id2 蛋白對於細胞週期所產生之影響(負調控)可經由 GCIP 而反轉-對於細胞週期調控蛋白E2F的影響41
討論.........................................43
參考文獻.....................................49
表...........................................53
圖...........................................54
附錄.........................................68
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