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研究生:李元皓
研究生(外文):Yuan-Hao Lee
論文名稱:利用切絲蛋白過度表現引發類老化現象之探討
論文名稱(外文):Induction of a Senescence-like Phenomenon by Over-expression of Cofilin
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:2009
畢業學年度:97
語文別:英文
論文頁數:95
中文關鍵詞:切絲蛋白p27Kip1G1週期停滯細胞老化
外文關鍵詞:Cofilinp27Kip1G1 phase arrestsenescence
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中文摘要
細胞老化阻止細胞無限制地生長分裂並促使細胞凋亡,這個現象卻無法在一些缺乏p53 和 p16INK4 抑癌蛋白的癌細胞上自然地發生。在老化的過程中,細胞會變得扁平而寬大,這外觀上的改變提供了我們一個重要訊息— 細胞骨架的改變或許跟細胞老化有關。因此,本篇研究係探討破壞細胞骨架肌動蛋白的組織結構是否會促使癌細胞產生老化現象。
目前已有研究顯示,肌動蛋白單體在細胞核的累積和細胞老化有關。又切絲蛋白負責細胞骨架肌動蛋白的去聚合作用與促進肌動蛋白在細胞核之分佈。因此,我們藉由改變切絲蛋白的表現量來探討細胞老化是否會受到影響。 HCOXP (H1299 with inducible cofilin over-expression) 細胞株帶有tet-on 基因表現的系統,可以被doxycycline誘發而使其切絲蛋白之過度表現。切絲蛋白過度表現造成肌動蛋白累積在細胞核內,其誘發之細胞老化現象則以SA β-gal (senescence-associated β-galactosidase) assay來評估。由實驗結果得知,切絲蛋白大量表現的細胞其β-gal呈色現象較為明顯。並且,切絲蛋白之過度表現引發了p27Kip1-mediated G1 phase arrest。在利用siRNA抑制細胞的p27Kip1表現後,細胞週期停滯現象解除了,且細胞的類老化現象也消失了。更進一步地,我們藉由光學測量法來定量出細胞類老化現象的程度並且發現此現象之發生與p27Kip1之大量表現有關。這些結果顯示了cofilin-p27Kip1之調控路徑影響了SA β-gal所決定的老化現象,且這些結果或許能提供癌症治療一個新的策略。
Abstract
Cellular senescence can prevent unlimited cell proliferation and may prompt cellular death. However, this phenomenon does not spontaneously occur in tumor cells lacking p53 and p16INK4 tumor suppressor proteins. Cells tend to become flattened and enlarged during senescence. This morphological change implies that actin cytoskeletal alteration may be related to cellular senescence. Thus, this study focused on investigating whether enforcement of actin cytoskeletal disorganization will affect the senescence in tumor cells.
Nuclear accumulation of monomeric actins has been reported to be associated with cellular senescence. In cells, actin deoplymerizing factor (ADF)/cofilin is responsible for increasing actin monomers and promoting actin relocalization to nucleus. Here we explore if altered expression of ADF/cofilin (cofilin hereafter) will affect the occurrence of senescence. HCOXP (H1299 with inducible cofilin over-expression) stable cell line harbors a tet-on gene inducible system that is able to over-express cofilin responding to tetracycline. Over-expression of cofilin led to accumulation of actin in nucleus. The extent of cellular senescence was determined by senescence-associated ��-galactosidase (SA-��-gal) staining, and the results showed that heavy staining of SA-��-gal was visualized in cofilin over-expressing cells. Concomitantly, over-expression of cofilin induced a p27kip1-mediated G1 phase arrest. Knockdown of p27kip1 expression by siRNA in cofilin over-expressing cells rescued the G1 phase arrest and reduced the level of the SA-��-gal staining. Furthermore, the extent of SA-�n��-gal staining was quantified by a spectroscopic system to confirm that over-expressed cofilin may promote a senescence-like phenomenon associating with up-regulation of p27kip1. These results suggest that a cofilin-p27kip1 regulatory pathway may be involved in the senescence phenomenon determined by SA-��-gal biomarker, and it may be an important implication in considering the therapeutic design for cancer treatment.
中文摘要 1
Abstract 2
Purpose 4
Chapter 1 Introduction 5
1.1 Senescence and its theory 5
1.2 Cellular senescence and cancer 6
1.3 Actin depolymerizing factor (ADF), cofilin 7
1.4 Tumor suppressor, p27Kip1 7
1.5 Spectroscopy for senescence-like phenomenon of β-galactosidase 8
Chapter 2 Materials and Methods 10
2.1 Cell cultures 10
2.2 siRNA transfection 10
2.3 Lentiviral infection using shRNA constructs 11
2.4 RT-PCR analysis 12
2.5 Flow cytometry and cell cycle analysis 13
2.6 Generation of cell growth curve 13
2.7 Western blot assay 14
2.8 Protein degradation analysis using cycloheximide 15
2.9 SA ß-gal assay 15
2.10 Spectroscopic measurement 16
2.11 Immunofluorescence microscopy 16
2.12 Trypan blue assay 17
2.13 Colony formation assay 17
2.14 Letiviral infection evaluated by EYFP expression 18
2.15 Statistic method 18
Chapter 3 Results 19
3.1 Cofilin over-expression led actin to accumulate in the nuclei of senescence-like HCOXP cells 19
3.2 Cofilin-induced G1 phase arrest was followed by cell growth retardation and cell survival reduction 20
3.3 P27Kip1 was up-regulated by cofilin over-expression in HCOXP cells and prompted to accumulate in the nuclei 20
3.4 Cofilin-induced G1 phase arrest and senescence-like phenomenon was rescued by p27Kip1 knockdown 21
3.5 Variances of SA ß-gal levels could be discriminated in HCOXP cells using spectroscopy 22
3.6 Cofilin-induced senescence-like phenomenon was not compromised by a lentiviral system 23
Chapter 4 Discussion 25
Chapter 5 Conclusion 28
References 29
Table 37
Figures 38
Appendix 65
1. Plasmid information for lentiviral production 65
2. Matlab codes for spectrometric analysis 67
3. Experimental techniques 72
4. Material information 79
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