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研究生:蘇伊平
研究生(外文):Yi-Ping Su
論文名稱:熱處理纖維母細胞之蛋白體分析
論文名稱(外文):Study of proteomic profiles of fibroblast in response to heat stimulation
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:82
中文關鍵詞:蛋白質體熱處理
外文關鍵詞:proteomicsheat shock
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熱處理(Heat shock)為外界刺激對細胞造成壓力的來源之一,過去的研究發現,高溫的刺激會造成細胞死亡,但若處以一個溫和的熱刺激反而會使細胞的活性增加,截至目前為止,是甚麼原因造成兩者的不同,仍有待進一步的研究工作進行探討。本實驗室之前為建立探討燒燙傷之細胞模型,以人類纖維母細胞作為研究材料,處理不同程度的溫度刺激人類纖維母細胞,發現以42℃處理時,能夠刺激細胞活性上升,反之,在52℃時的處理則讓細胞走向死亡。
本研究目的在於利用蛋白質體學的方法,建立燒燙傷細胞模型之蛋白質體資料庫,同時輔以生物資訊學的分析方法,探討在不同溫度處理下,重要蛋白質之表現量變化與訊息傳導路徑之改變,從中歸納出不同溫度造成細胞不同反應的可能原因。
首先以二維電泳展開37℃、42℃、52℃處理下之蛋白質表現圖譜,從中鑑定出35個表現量有差異的蛋白質,根據生物資訊學的分析結果,我們將蛋白質的表現變化分為三個群組,分別代表不同的生物意義。其中最大的差別之一在於,42℃的處理狀況下,六個具有抗氧化活性的蛋白質表現量增加,這顯示在熱刺激下細胞內的Reactive oxygen species(ROS)對細胞存活可能扮演相當關鍵的角色。從DCFDA偵測結果可以看到,熱刺激的確會造成細胞內ROS的上升,進一步從西方墨點測試來進一步確認PRDX2、PARK7兩個具抗氧化功能蛋白的表現量,同時探討ROS調控之相關訊息傳導路徑,我們看到在52℃處理的
狀況下,JNK的訊息傳遞路徑有明顯的被活化,反之,ERK的活化則不顯著。因此我們認為,決定熱處理後人類纖維母細胞走向存活或死亡的關鍵因素之一在於,ROS所調控的訊息傳導路徑變化。
Heat shock response has been found to influence cell viability since three decades
ago. Mile heat stress result in cell survival and proliferation while sever heat stress
lead to cell death. However, the issue of mechanisms or pathways contribute to such
different kinds of cell response under heat shock remains to be elucidated.
Previous results showed increase of cell viability occurs at 42℃ while incubation
fo the cells at 52℃ induces cell death. To reveal the cause of cell survival, we
analyzed the difference of proteome changes of cells under various of heat shock
treatments. Proteomic as well as bioinformatic approach were adapted to investigate
the protein profile changes with heat treatment at 37℃, 42℃, and at 52℃ . The
spectrometric analysis of the protein spots from 2-D PAGE indicated that 35 protein
spots showed significant differentially expressed. Using hierarchical clustering
analysis, these proteins were categorized into three major expression patterns.
Interestingly, a group of antioxidant proteins responsed only to mild heat shock,
rather than sever heat shock. Hence, detection of the ROS level of cells under heat
shock indicated that ROS may play a role as a modulator in determining cell fate
under heat shock. Moreover, the western blot analysis of cells under heat shock
showed that JNK and ERK signal pathways may be involved in the cell viability
during heat shock treatment. Taken together, our results may shed light on the fates of
cells in response to heat treatment.
英文摘要-------------------------------------------------------------------------------------------------------------------------------------1
中文摘要 ------------------------------------------------------------------------------------------------------------------------------------2
第一章、緒論 ----------------------------------------------------------------------------------------------------------------------------4
一、皮膚結構與組成-----------------------------------------------------------------------------------------------------------------4
二、傷口癒合過程---------------------------------------------------------------------------------------------------------------------5
三、人類纖維母細胞之角色-----------------------------------------------------------------------------------------------------6
四、熱處理反應(Heat Shock Response)-------------------------------------------------------------------------------6
五、溫和熱處理與嚴重熱處理--------------------------------------------------------------------------------------------------8
六、熱處理與細胞膜之生理特性----------------------------------------------------------------------------------------------9
七、MAPK訊息傳導路徑----------------------------------------------------------------------------------------------------------9
八、熱休克蛋白與MAPK--------------------------------------------------------------------------------------------------------12
九、研究動機與目的---------------------------------------------------------------------------------------------------------------12
第二章、實驗方法------------------------------------------------------------------------------------------------------------------13
一、人類纖維母細胞培養-------------------------------------------------------------------------------------------------------13
二、細胞熱處理方法---------------------------------------------------------------------------------------------------------------14
三、蛋白質萃取及含量測定----------------------------------------------------------------------------------------------------15
四、二維蛋白質電泳分析-------------------------------------------------------------------------------------------------------17
五、二維蛋白質電泳膠片染色------------------------------------------------------------------------------------------------19
六、二維蛋白質電泳軟體比對分析----------------------------------------------------------------------------------------20
七、In-gel digestion------------------------------------------------------------------------------------------------------------------20
八、利用質譜儀進行蛋白質身分鑑定------------------------------------------------------------------------------------21
九、生物資訊學分析方法-------------------------------------------------------------------------------------------------------22
十、ROS assay-------------------------------------------------------------------------------------------------------------------------22
十一、 SDS-PAGE蛋白質電泳分析----------------------------------------------------------------------------------------23
十二、西方墨點轉漬法-----------------------------------------------------------------------------------------------------------23
I
第三章、實驗結果------------------------------------------------------------------------------------------------------------------25
一、蛋白質圖譜分析與階層式分群法分析----------------------------------------------------------------------------25
二、蛋白質生物性功能分類分析--------------------------------------------------------------------------------------------26
三、2' , 7' -dichlorofluorescin diacetate探測活性氧化化物(ROS)---------------------------------------28
四、訊息傳遞路徑之偵測-------------------------------------------------------------------------------------------------------29
第四章、討論---------------------------------------------------------------------------------------------------------------------------32
一、 Stathmin角色之探討--------------------------------------------------------------------------------------------------------32
二、多聚胺(Polyamine)角色之探討-----------------------------------------------------------------------------------33
三、熱處理下活性氧分子(ROS)角色之探討---------------------------------------------------------------------34
四、熱處理下Mitogen-activated protein kinase家族之功能探討---------------------------------------------36
五、熱休克蛋白與MAPK家族------------------------------------------------------------------------------------------------38
六、熱處理與細胞骨架(Cytoskeleton)-------------------------------------------------------------------------------38
七、熱處理與泛素化作用(Ubiquitination)-------------------------------------------------------------------------39
八、熱處理與細胞運動性(Cell Motility)----------------------------------------------------------------------------40
九、總結---------------------------------------------------------------------------------------------------------------------------------40
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