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研究生:林明慧
研究生(外文):Ming-Hui Lin
論文名稱:v-Src轉化細胞中表現量下降的蛋白質Secernin1之研究
論文名稱(外文):Characterization of Secernin 1, a protein down-regulated in v-Src transformed cells
指導教授:林照雄林照雄引用關係
指導教授(外文):Chao-Hsiung Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物藥學研究所
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:2006
畢業學年度:94
語文別:中文
論文頁數:59
中文關鍵詞:RK3E/tv-a/Src細胞外釋作用v-Src誘發轉化轉移
外文關鍵詞:RK3E/tv-a/Src cellsexocytosisin v-Src-transformed cellsmatastasis
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傳醫所傅淑玲老師的研究室在觀察RK3E/tv-a細胞與RK3E/tv-a/Src細胞的蛋白質表現差異時,觀察到一個表達量下降的未知蛋白,經過序列比對之後發現這個蛋白質與小鼠的Secernin 1蛋白質有高度同源性,此外利用RT-PCR的實驗亦可觀察到Secernin 1的mRNA表現在v-Src 轉化細胞中也有減少的情形,因此在本論文中想要進一步地探討Secernin 1在v-Src轉化細胞中所扮演的功能。Secernin 1最初是在2002年由Antony等人發現它在肥胖細胞中具有可以調控外釋作用(exocytosis)的能力,另外最近Tahara等人在2006年也發現Secernin 1可以作為一個新穎胃癌細胞的immunotherapy target。除此之外,有關Secernin 1的研究報告付之闕如,因此我們進行了一些生物資訊的搜尋,發現在NCI 60白血病細胞株中,Secernin 1有明顯被抑制的情形;在HUGE database的北方墨點法結果裡亦發現Secernin 1在各種組織中都存在,但以大腦中含量最高;此外,在蛋白質結構預測方面,Pfam資料庫將Secernin 1歸屬於peptidase_C69 family之中。在本論文之中我們將報告有關於Secernin 1在v-Src誘導轉化中的功能探討及它的過度表現對轉化影響的研究進展。目前我已成功地將Secernin 1表現在大腸桿菌中及進一步地分離出純化蛋白質,並已獲得可以進行免疫偵測的多株抗體;而在探討Secernin 1與v-Src及其他致癌基因之間的關係中,發現在有Src,Akt和Ras等基因存在細胞時,Secernin 1均被觀察到表達量下降的趨勢。但是在temperature-inducible v-Src 轉化細胞中卻沒有下降的情形,故推測Secernin 1與v-Src誘發轉化的過程中沒有直接影響的關係。另外在具有轉移能力的v-Src 轉化細胞中也有觀察到Secernin 1的表現持續下降的趨勢,但是進一步在v-Src 轉化細胞中過量表現Secernin 1時,並不會影響該細胞migration和invasion的能力。另外在三對不同轉移能力的細胞株對照中並沒有看到Secernin 1的表現量下降。綜合我的實驗結果,Secernin 1在v-Src轉化細胞以及具有轉移能力的細胞中有表現下降的趨勢,但推測與Src沒有直接的關係,且在轉移的過程中並非必要的影響因子,因此,Secernin 1在Src轉化細胞的功能角色仍需要再更深入的探討與研究。
Using proteomic approach, Dr. Shu-ling Fu’s laboratory at Institute of Traditional Medicine, National Yang-Ming University has compared the proteomes of RK3E/tv-a and RK3E/tv-a/Src cells and identified a down-regulated protein in v-Src-transformed cells. This protein shows high homology to mouse Secernin 1. Using RT-PCR, Dr. Fu observed that mRNA expression of Secernin 1 in v-Src-transformed cells was also decreased. The goal of this thesis is to characterize Secernin 1 in v-Src-transformed cells. In 2002, Antony et al. discovered that Secernin 1 regulates exocytosis in mast cells; Recently, Tahara et al. also identified Secernin 1 as a novel immunotherapy target for gastric cancer. However, there is no other literature regarding the functional role of Secernin 1. During the preliminary bioinfomatics search for background information of Secernin 1, we found that the expression of Secernin 1 decreases in leukemia cancer cell line of NCI60. According to the Northern blot analysis in HUGE database, Secernin 1 ubiquitously expressed in all tissues, yet showing the highest expression in brain. Protein structure analysis using Pfam program identifies Secernin 1 as a member of peptidase_C69 family. In this dissertation, I reported the studies to characterize the functional role of Secernin 1 in v-Src transformation and to determine whether overexpression of Secernin 1 is able to influence phenotypes of v-Src transformed cells. I successfully expressed and purified recombinant Secernin 1 from E.coli, and developped a polyclonal antibody against Secernin 1. During the functional studies of Secernin 1 with different oncogenes, I observed that expression of Secernin 1 decreased in cells overexpressing Src, Akt, and Ras cells. However, its expression retains unchanged in temperature-inducible Src-transformed cells. Therefore, we speculated that Secernin 1 is not necessarily required for v-Src transformation. In addition, we also observed that the expression of Secernin 1 further decreased in v-Src-transformed cells with metastatic feature, but overexpression of Secernin 1 in these cells did not affect cell migration and invasion. To summarize my results, expression of Secernin 1 was decreased in v-Src-transformed and its derived metastatic cells;We speculated that Secernin 1 did not have direct relationship with Src and is not a necessary factor in matastasis. Therefore, the functional roles of Secernin 1 in v-Src-transformed cells demands further studies.
摘要
英文摘要
縮寫表
壹、緒論
貳、材料與方法
參、結果
肆、討論
伍、參考文獻
陸、圖表
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