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研究生:林君翰
研究生(外文):Chun-Han Lin
論文名稱:探討纖維母細胞耐熱性之蛋白體研究
論文名稱(外文):The Study of Thermotolerance in Fibroblast Revealed by Proteomic Approach
指導教授:翟建富翟建富引用關係
指導教授(外文):Kin-Fu Chak
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:76
中文關鍵詞:蛋白質體熱處理耐熱性
外文關鍵詞:proteomic analysisheat shockthermotolerance
相關次數:
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摘要
耐熱性是指給予細胞一個溫和的刺激後,能誘導細胞產生對於接下來更高溫度的抗性。普遍認為耐熱性與heat shock protein有密切的關係,然而耐熱性的產生並非單一因素所能造成的,目前對於他的詳細機制未能有明確通盤的解釋。
纖維母細胞存在於皮膚真皮以及皮下組織,能維持細胞間質,分泌激素,是皮膚傷口修復的主要角色,是燒燙傷研究中的主要材料。本篇研究目的是探討耐熱性在纖維母細胞中被誘導出來的原因及其分子生物學上的機理。
藉西方墨點的測詴得知,纖維母細胞中的耐熱性可能與MAPK中JNK路徑的調控有關,耐熱性能抑制JNK路徑的活化,從而阻止細胞凋零的進行。在蛋白質體學的結果中,利用生物資訊學將功能相似的蛋白體類分成抗氧化,促細胞增生,離子恆定調控等相關蛋白體,從自由基與鈣離子的相關測詴中發現,耐熱性能緩衝自由基的產生,以及能穩定細胞內鈣離子的濃度。
我們的實驗結果建立了耐熱性產生的分子生物學機理。耐熱性不會是單一因素所誘導出的結果,而是細胞內各因子通力合作所獲得對環境適應力的結果。
Abstract
Thermotolerance is the protection ability that cell pre-treated with a mild heat shock can survive in a following severe stress. It was believed that thermotolerance is closely correlated to heat shock response, however, becoming clearer that thermotolerance is induced by multiple factors instead of a single element. Thus far, the comprehensive mechanism of thermotolerance is still remained to be resolved.
In response to different environment stresses, each type of cell has diverse physiological process to adapt itself to environment challenge. The fibroblast is the main cell type of skin. It can maintain extracellular matrix and secrete growth factor to help wound recovery. It is therefore fibroblast is becoming the most important tool for the study of wound burn healing processes. Thus, the aim of the study is to reveal the fundamental molecular basis of the acquired thermotolerance of the fibroblast.
According to the results of Western blot analysis, the thermotolerance can inhibit the activation of JNK pathway, leading to the reduction of apoptosis of the cells. Using BGSSJ for bio-information analysis, proteins detected by proteomic analysis have been clustering into different functional groups including anti-oxidant, promotion of proliferation, and maintenance of ion flux, etc. Our reactive oxygen species and calcium assay implied that occurrence of thermotolerance may buffer the generation of free radical and keep ion flux steady.
In this study, we have provided a comprehensive viewpoint in molecular basis for the occurrence of thermotolerance and that thermotolerance is not an effect induced by a single factor but an adaptive ability came from a set of teamwork of multiple factors in cell.
目錄 1
Abstract 4
摘要 6
緒論 7
一、 背景介紹 7
1. 細胞vs.環境壓力(Cell vs. Stress) 7
2. 耐熱性(Thermotolerance) 8
3. 信號傳遞的調控(Signaling transduction pathway) 9
4. 氧化壓力(Oxidative stress) 10
5. 離子恆定(Regulation of ion flux steady) 11
6. 細胞凋零(Apoptpsis) 12
7. 蛋白質體分析(Proteomic analysis) 13
二、 研究動機以及研究策略 15
1. 研究動機 15
2. 研究策略 15
實驗材料與方法 17
一、 纖維母細胞培養 17
二、 細胞熱處理方法 (Heat shock) 19
三、 細胞內蛋白質萃取以及定量 20
四、 SDS-PAGE蛋白質電泳分析 21
五、 西方墨點測試 22
六、 二維蛋白質電泳分析 23
七、 快速銀染法 24
八、 In-gel digestion 25
九、 蛋白質體軟體分析 27
十、 ROS assay 28
十一、 Intracellular calcium assay 28
實驗結果 29
一、 耐熱性的信號路徑啟動探討 29
二、 以二維電泳分析耐熱性誘導表現之蛋白 30
三、 活性氧化物含量分析 32
四、 鈣離子含量分析 33
結果討論 35
一、 JNK路徑磷酸化的西方墨點結果分析 35
二、 二維電泳蛋白質體分析討論 36
三、 細胞內活性氧化物濃度結果分析 49
四、 細胞內鈣離子濃度結果分析 50
五、 結論 51
參考文獻 54
附圖 61
圖一: 61
圖二: 62
圖三: 63
圖四: 64
圖五: 65
圖六: 66
圖七: 67
圖八: 68
圖九: 69
圖十: 70
表一之一: 71
表一之二: 72
表二: 73
附錄一: 74
附錄二: 75
附錄三: 76
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