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研究生:江佩珣
研究生(外文):Pei-Hsun Chiang
論文名稱:探討切絲蛋白於細胞週期進行時之表現量變化與細胞放射敏感度之關聯性
論文名稱(外文):Association of actin-regulation protein cofilin on cell cycle progression and radiosensitivity
指導教授:李易展
指導教授(外文):Yi-Jang Lee
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物醫學影像暨放射科學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:83
中文關鍵詞:切絲蛋白細胞週期游離輻射輻射敏感度DNA修復
外文關鍵詞:CofilinCell cycleIonizing radiationRadiosensitityDNA repair
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微絲骨架(Actin filament)為構成細胞整體結構與維持形狀的重要蛋白。許多研究指出,細胞週期的進行與微絲骨架的動態調節具有緊密的相關性,而微絲骨架主要藉由聚合與去聚合作用調節微絲骨架動態的變化。切絲蛋白 (Cofilin)為微絲去聚合分子家族 (Actin depolymerization factor)成員之一,藉由去聚合特性調節微絲整體的動態性。過去研究指出大量表現切絲蛋白將會造成細胞週期G1停滯,本研究將探討切絲蛋白於細胞週期進行下的變化,推測在G1時期,切絲蛋白表現將為最低量。另一方面,細胞週期的改變與輻射敏感度息息相關,當細胞經游離輻射曝射後,造成DNA雙股斷裂,使細胞走向死亡。過去文獻指出,大量表現切絲蛋白會增加細胞的輻射敏感度,推測切絲蛋白將影響DNA修復的能力,其中機制仍待釐清。
肺癌為全球發病率和死亡率極高的癌症之一,本次實驗中以人類非小細胞肺癌細胞 A549 (p53野生)與H1299 (p53缺失)細胞株探討細胞週期進行下切絲蛋白的表現。將細胞同步化於各細胞週期,發現微絲骨架結構與切絲蛋白的表現將隨著所處週期不同而有所變動,當兩株細胞處於G1時期時,切絲蛋白表現均遠低於S時期。為了進一步探討切絲蛋白與DNA修復之間的關係,我們利用可大量表現人類切絲蛋白基因之穩定轉殖人類肺癌HCOXP細胞株(H1299 with cofilin over- expression)。利用此細胞誘發大量表現切絲蛋白後,抑制γH2AX的形成,並影響下游同源重組 Rad51與非同源黏合修復路徑中 Ku70與Ku80修復蛋白的表現,造成DNA修復的延緩進而增加細胞的輻射敏感度。
在本研究中證實兩株人類肺癌細胞的切絲蛋白表現隨細胞週期進行而有所變化,且G1時期的表現均低於S時期。此外,大量表現切絲蛋白將影響此平衡造成G1時期停滯,更進一步的證實大量表現切絲蛋白可抑低DNA修復能力,進而增加輻射敏感度。提供未來以切絲蛋白作為標靶,影響細胞週期與輻射敏感度的治療策略。

Actin filament is the most abundant protein in eukaryotic cells and essential for maintaining the cellular shape and structure. Several lines of evidence had demonstrated that alteration of the cell cycle distribution by interferance of actin filament. Cofilin is a member of actin depolymerizing factor family that can regulate actin dynamic and severance of actin filament. In this study, we investigated the role of cofilin within cell cycle regulation. Several lines of evidence have demonstrated that cofilin is associated with the cell cycle regulation and over-expression of cofilin led to G1 arrest. We speculated the expression of cofilin should be the lowest in G1 phase. On the other hand, the radiosensitivity is highly dependent on cell cycle. Ionizing radiation would cause DNA double strand breaks and result in cell death. Previous study showed that over-expression of cofilin increased radiosensitivity. Thus, we hypothesize that cofilin plays a crucial role in DNA repair capacity.

Lung cancer is one of the most common cancers in the world. In this study, we used two human non-small cell lung cancer cell lines, A549 (p53 wt) and H1299 (p53 null), for investigation of the relationship between cofilin and cell cycle progression. We dissected the cofilin expression in each phase of the cell cycle. It was found that the protein level of cofilin was fluctuated during cell cycle progression, and the lowest expression of cofilin was appeared in G1 phase, instead of others., To better understand the role of cofilin in DNA repair capacity, we applied the HCOXP cell line (H1299 cofilin over-expression) to over-expressed cofilin via tetracycline-inducible system. We found that overexpression of cofilin inhibited H2AX phophorylation through ATM pathway, resulted in reduction of NHEJ and HRR repair factor recruiment
These findings suggest that precise regulation of cofilin expression is important for cell cycle progression and further reveal that over-expression of cofiln could reduse DNA repair capacity. In coclusion, cofilin is an attractive target to regulate cell cycle and enhance radiosensitivity.

中文摘要...................................................1
Abstract...................................................3
壹.緒論....................................................5
一.細胞微絲骨架 (actin filamemt,microfilament)............5
二 微絲骨架去聚合分子,切絲蛋白 (Cofilin) .................6
三.細胞週期調控 --- Cyclin/CDK複合體 與CDKI表現............8
四.細胞週期調控與微絲骨架之關係............................9
五. 細胞週期與輻射效應....................................10
六.DNA 雙股斷裂與修復途徑.................................11
貳.研究動機...............................................13
叁.材料方法...............................................14
一.細胞培養...............................................14
二.細胞週期同步化 (Synchronizaiotn).......................14
三.細胞週期分佈之分析.....................................15
四.西方墨點轉漬法(蛋白質電泳).............................16
五.免疫螢光染色法 (Immunofluorescence)....................19
六.DNA 單股斷裂偵測法 (Comer assay).......................20
七.細胞存活曲線分析 (Survival curve / Colony formation assay) ...................................................21
八.shRNA 慢病毒感染抑低表現系統...........................21
九. RNA萃取與Reverse-transcription PCR....................22
十.輻射源.................................................22
十一.統計分析.............................................22
肆.研究結果...............................................24
一.切絲蛋白與細胞週期進行之關.............................24
I.各細胞週期下細胞微絲骨架分佈變..........................24
II.利用各同步化方式觀察微絲骨架切絲蛋白Cofilin於各細胞週期表現........................................................24
III.細胞週期進行下切絲蛋白表現變化........................25
IV.大量表現切絲蛋白將改變微絲骨架結構與細胞週期分佈.......25
二.切絲蛋白與輻射敏感度之關係.............................29
I.大量表現切絲蛋白並照射游離輻射將造成大量DNA單股及雙股的斷裂........................................................29
II.大表現切絲蛋白經游離輻射曝射後明顯增加輻射敏感度.......30
III.經游離輻射曝射後,DNA 雙股斷裂修補蛋白變化............30
IV.大量表現切絲蛋白將會影響DNA受損之訊號傳遞..............31
三.切絲蛋白藉調節p27kip1蛋白影響細胞週期的進行與DNA 修復蛋白的基礎表現................................................33
伍.討論...................................................34
陸.結論:..................................................41
柒.參考文獻...............................................42
捌.附圖...................................................49
玖.附錄...................................................70
附錄一....................................................70
附錄二....................................................71
附錄三....................................................72
附錄四....................................................73
附錄五....................................................74
附錄六....................................................75
附錄七....................................................77
附錄八....................................................77



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