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研究生:陳語農
研究生(外文):Yu-nong Chen
論文名稱:過度表現微小球蛋白MSP58誘導細胞老化機制之探討
論文名稱(外文):Overexpression of the Microspherule Protein 58(MSP58) gene induces senescence-like state in HT1080 cells
指導教授:林鼎晏張文昌張文昌引用關係
指導教授(外文):Ding-yen LinWen-chang Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
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:77
中文關鍵詞:端粒酶衰老
外文關鍵詞:SenescenceTelomerase
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端粒酶活性與細胞增生、分化、細胞老化及癌症形成息息相關,其中因為端粒酶活性與細胞老化的相關性讓許多學者有興趣去探討彼此的關聯。研究發現微小球蛋白MSP58可能參與在細胞轉型的機制中,而被認為是一種致癌基因的角色。然而有文獻指出微小球蛋白MSP58的isoform,MCRS2蛋白,能與抑制端粒酶活性的蛋白LPTS/PinX1互相結合,並且過度表現MCRS2蛋白會導致端粒縮短及抑制端粒酶活性。在此論文中我們發現過度表現微小球蛋白MSP58會導致細胞產生類似衰老的狀態。研究過程中我們將微小球蛋白MSP58持續表現在HT1080細胞中,結果顯示過度表現的微小球蛋白MSP58會同時延遲G1/S期的轉換,及細胞分裂期到G1期之間的過程,另外也促使細胞產生衰老的型態與生化特性,包括了細胞體積增大且扁平,減少攝入BrdU的速率,增加senescence-associated β-galactosidase的活性及抑制端粒酶活性。在機制探討部分,我們發現生長及細胞週期抑制的原因,有可能是透過 pRb的磷酸化比例下降,與cyclin-dependent kinase inhibitors p21的蛋白表現量提升,這兩個機制所造成。根據我們的結果推論, 微小球蛋白MSP58可以經由調節pRb與p21相關的訊息傳遞導致類似衰老細胞的生長抑制現象。
The 58-kDa microspherule protein (MSP58; MCRS1) was initially shown to interact with p120, a proliferation-related protein expressed at high levels in most human malignant tumor cells. A study of the quail homologue of MSP58, TOJ3, showed that this protein exhibits cell transformation activity. Recently, a study revealed that MSP58 behaves as an oncogene and that its transformation activity can be inhibited by physical interaction with PTEN tumor suppressor. Moreover, a splice isoform of human MSP58, MCRS2, was found to interact with the potent telomerase inhibitor LPTS/PinX1. Here, we shown that overexpression of the MSP58 gene induces senescence-like state in cells. Studies in the MSP58 stably expressed clones in HT1080 cells revealed that overexpression of MSP58 inhibited both G1/S progression and delay progression from mitosis to G1 and conferred morphological and biochemical features of senescence, including cells exhibited enlarged and flattened, reduced BrdU incorporation rates, elevated senescence-associated β-galactosidase activity and reduced telomerase activity. Mechanistic analysis revealed that the arrest may in part be accounded for by inducing hypophosphorylation of pRB and up-regulation cyclin-dependent kinase inhibitors p21. Our results suggest that MSP58 control cellular proliferation and senescence by modulating the pRb- and p21- related pathways.
中文摘要................................................Ⅰ
英文摘要................................................Ⅱ
誌謝....................................................Ⅲ
目錄....................................................Ⅳ
圖目錄..................................................Ⅶ
附錄....................................................Ⅷ
第一章 緒論..............................................1
第一節 細胞衰老......................................2
第二節 端粒與端粒酶..................................3
第三節 端粒與端粒酶 vs. 衰老及癌化...................4
第四節 細胞週期與細胞衰老............................5
第五節 Microspherule Protein 58(MSP58)之介紹.........7
第六節 研究動機......................................8
第二章 實驗方法與材料....................................9
第一節 人類纖維肉瘤細胞株HT1080之培養................9
第二節 質體之建構...................................10
第三節 持續表現MSP58蛋白質細胞株之製備..............14
第四節 全細胞液之抽取與蛋白質定量...................15
第五節 硫酸十二酯鈉聚丙烯醯胺凝膠電泳法(SDS-PAGE)/西方墨點
法(Western blot).............................17
第六節 免疫螢光染色法(Immunofluorescence)...........23
第七節 利用細胞計數及BrdU的嵌入觀察細胞生長.........25
第八節 細胞同步化與細胞週期分析.....................28
第九節 流式細胞儀(Flow cytometry)分析...............31
第十節 端粒重複序列放大反應(Telomere Repeat Amplification
Protocol, TRAP assay) .......................32
第十一節 由Senescence associated β-galactosidase (SA-β-gal)之活性
測試反映細胞衰老程度....................................37
第三章 實驗結果........................................39
第一節 確認Microspherule Protein 58(MSP58)
的質體在HT1080細胞模式中的表現..........................39
第二節 在HT1080細胞中過度表現MSP58
蛋白會抑制細胞生長及細胞增生............................39
第三節 在HT1080細胞中過度表現MSP58蛋
白會導致類似衰老的細胞型態改變..........................40
第四節 在HT1080細胞中過度表現MSP58
蛋白會延遲細胞週期的進行................................41
第五節 在HT1080細胞中過度表現MSP58
蛋白會抑制端粒酶的活性..................................43
第六節 在HT1080細胞中過度表現MSP58蛋白使細胞呈現
Senescence associated β-galactosidase活性...44
第四章 討論.............................................45
第五章 參考文獻.........................................49
自述....................................................77
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