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研究生:陳哲鑫
研究生(外文):Je-Hsin Chen
論文名稱:影響蛋白磷酸激酵素C活性因子對金屬硫蛋白基因表現的研究
論文名稱(外文):Effect of factors involved in protein kinase C activity on metallothioneingene expression
指導教授:林立元林立元引用關係
指導教授(外文):Lih-Yuan Lin
學位類別:博士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2001
畢業學年度:90
語文別:中文
中文關鍵詞:金屬硫蛋白蛋白磷酸激酵素C中國倉鼠卵巢細胞株
外文關鍵詞:metallothioneinprotein kinase CcadmiumCHO cellrottlerin
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摘要
金屬硫蛋白(MT)為普遍存在於生物細胞內的低分子量蛋白質,其特性為具有高金屬(Zn、Cu、Cd)結合能力、高半胱胺酸(cysteine)含量。MT基因很容易被金屬、化學物質或逆境誘導而表現。為了明瞭MT基因被金屬誘導的機制,因此進行以下研究:1)以訊號傳遞路徑中蛋白磷酸激酵素或磷酸水解酵素的抑制劑來篩檢金屬誘導MT基因表現可能包含的訊號傳遞路徑。2)以影響細胞中氧化還原物質含量的藥劑處理細胞來推測金屬誘導MT基因表現的路徑是否包含產生反應性氧種類(reactive oxygen species; ROS)。3)由於研究過程中發現rottlerin不但不能抑制金屬誘發MT基因的表現,本身反而增加MT基因的表現。因而進而探討其誘導MT基因表現可能的機制。
以訊號傳遞路徑中磷酸激酵素或磷酸水解酵素的抑制劑處理細胞,用北方吸漬法來檢驗是否影響金屬誘導MT基因表現,結果發現大部份磷酸激酵素抑制劑均無法影響金屬誘導MT基因表現。其中包括了classical PKC的抑制劑(HBDDE, Gö6976)、磷脂質水解酵素抑制劑(neomycin, RHC-80267, U-73122)、calmodulin抑制劑(W7)、蛋白tyrosine磷酸激酵素抑制劑(herbimycin A)、與MEK1抑制劑(PD98059)。能夠抑制金屬誘導MT基因表現的僅有廣效性PKC抑制劑H7,而磷脂質水解酵素(PL-A2)抑制劑manoalide和蛋白磷酸水解酵素(PP-2A, PP1)抑制劑okadaic acid則僅有部份抑制的效果。PKCd抑制劑rottlerin和蛋白tyrosine磷酸水解酵素抑制劑vanadate則促進金屬對MT基因的誘導。
本研究以過氧化氫處理細胞來增加細胞中ROS的含量、以DL-buthionine-[S, R]-sulfoximine (BSO)來處理抑制細胞中GSH的生成,或以ascorbic acid、a-tocopherol處理細胞做為ROS的清除劑,結果顯示,這些外加處理均不影響金屬對MT基因的誘導。而N-acetyl-L-cysteine (NAC)處理來增加細胞中GSH含量或3-amino-1, 2, 4-triazole (3-AT)處理來抑制catalase活性均使金屬對MT基因的誘導降低。由於3-AT前處理會抑制金屬在CdR細胞中的累積含量,而NAC具有可與金屬相結合的SH基團,因此3-AT和NAC抑制金屬對MT基因的誘導可能是因為抑制金屬進入細胞的關係。以mannitol做為細胞中氫氧自由基的清除劑則只能部份抑制Cd對MT基因的誘導,無法抑制Zn對MT基因的誘導。因此在CdR細胞中金屬對MT基因誘導的訊號傳遞路徑中,是否包含尚未發現的磷酸化路徑或包含產生ROS的活化路徑仍需要進一步探討與釐清。
參與對PKC異構酵素具專一性的抑制劑來篩選金屬誘導MT基因可能的PKC種類。實驗結果顯示,以北方吸漬法或RT-PCR的方法證明,在CdR細胞中PKCd的抑制劑-rottlerin非但不能抑制MT基因的表現,反而誘發此基因的轉錄。進一步分析此誘導機制得知,rottlerin處理細胞並不能有效增加MTF-1(MRE-binding transcription factor-1)蛋白質的含量,同時也抑制了金屬進入細胞中,但是卻活化MTF-1對MRE (metal response element)序列的結合活性。因此我們猜測rottlerin誘導MT基因的表現是由於造成細胞中Zn的重新分布,進而活化MTF-1的活性的結果。但是以大鼠腦下垂體腫瘤細胞GH3為材料所得結果,rottlerin並不能活化MTF-1的結合活性或增加MT基因被金屬誘導的表現,也無法抑制Cd進入細胞中。顯然rottlerin影響細胞中Zn的分布而活化MTF-1的結合活性及誘導MT基因的表現具有細胞專一性。
Abstract
Metallothionein(MT) is a high metal content, cysteine-rich low molecular weight protein ubiquitous in animal cells. Its expression can be induced by metals、chemicals or under various stresses. To understand the factors involved in the induction of MT protein expression by metals, in this study we have selected and characterized several potential inhibitors of protein kinase and protein phosphatase that may involve in signaling pathway of MT gene expression. We have also investigated whether reactive oxygen species (ROS) is involved in the pathway for activation of MT gene or not by using various chemicals influenced the redox-oxidation reaction in cells. We found that rottlerin, a PKCd specific inhibitor, can effectively activate the MT gene expression, and the mechanism of MT gene induced by rottlerin was also investigated in this study.
Result from Northern blot analysis showed that most of the protein kinase inhibitors exhibited no or little effect on the induction of MT gene expression by metals. Inhibitors have been tested in this study including classical PKC inhibitors (HBDDE, Gö6976), phospholipase inhibitors (neomycin, RHC-80267, U-73122), calmodulin inhibitor (W7), protein tyrosine kinase inhibitor (herbimycine A) and MEK1 inhibitor (PD98059). H7, a broad range PKC inhibitor, is the only inhibitor conferring inhibition activity on MT gene expression induced by metals. Manoalide, a phospholipase-A2 inhibitor, and okadaic acid, a protein phosphatase-2A and protein phosphatase-1 inhibitor, partially inhibited MT gene expression. However, rottlerin, a PKCd inhibitor, and vanadate, a tyrosine phosphatase, increase MT gene expression.
There were no effect detected on MT gene expression induced by metals when cells were pre-treated with hydrogen peroxide to increase ROS, DL-buthionine-[S, R]-sulfoximine (BSO) to inhibit synthesis of glutathione (GSH), or ascorbic acid or a-tocopherol to scavenge ROS. However, MT gene expression can be effectively inhibited by either N-acetyl-L-cysteine (NAC), a chemical elevates cellular GSH synthesis, or 3-AT, a catalase inhibitor. Although mannitol can act as a hydroxy radical scavenger, it just partial inhibits MT gene expression induced by cadmium, but not zinc ion. Whether the signaling pathway of metal induced MT gene expression in CdR cells involves in phosphorylation cascade or ROS synthesis is remain to be determined.
Rottlerin activated MT gene expression can be detected by Northern blot and RT-PCR. Although cells treated with rottlerin inhibited metal uptake into cells and did not increase cellular MRE-binding transcription factor-1 (MTF-1) content, metal responsive element (MRE) binding activity of MTF-1 was elevated. These results indicate that intracellular zinc ion is possibly re-distributed after cells were treated by rottlerin. However, no effect aws observed when GH3 cells were treated with rottlerin at the same condition. The results showed that rottlerin did not increase MTF-1 binding activity to MRE, whereas induced MT gene expression and inhibited Cd uptake. These results indicate that induction mechanism of MT gene expression by rottlerin is cell type specific.
目錄
一、緒論------------------------------------------------------1
A.金屬硫蛋白的結構-------------------------------------2
B.MT的功能---------------------------------------------5
細胞增生和細胞凋亡--------------------------------------------5
維持必需金屬的恆定--------------------------------------------7
扮演自由基的清除者--------------------------------------------8
金屬的解毒功能------------------------------------------------9
C.金屬硫蛋白基因的表現--------------------------------11
金屬硫蛋白的誘發劑-------------------------------------------11
D.金屬硫蛋白基因的構造--------------------------------16
E.高等真核細胞MT基因轉錄的調節------------------------17
F.PKC與訊號傳遞路徑的關係-----------------------------22
G.研究目的--------------------------------------------23
二、材料與方法-----------------------------------------------27
材料---------------------------------------------------------27
細胞的培養---------------------------------------------------27
pGEX-3KS-MTF 表現載體的構築----------------------------------27
雙股DNA基模的萃取--------------------------------------------28
DNA的定序反應------------------------------------------------29
DNA定序電泳--------------------------------------------------30
供轉形細胞的備製---------------------------------------------31
細胞的轉形---------------------------------------------------31
細胞質中RNA的提取--------------------------------------------31
RNA的電泳----------------------------------------------------32
北方吸漬法---------------------------------------------------33
逆轉錄酵素-聚合酵素鏈鎖反應(RT-PCR)--------------------------33
全細胞蛋白粗萃取液的提取-------------------------------------35
SDS-聚丙烯醯氨凝膠電泳---------------------------------------35
蛋白質的轉移-------------------------------------------------36
西方吸漬法---------------------------------------------------36
細胞中鎘和鋅含量的測定---------------------------------------36
EMSA---------------------------------------------------------37
放射性同位素S35短暫標記MT------------------------------------38
細胞族群存活率分析-------------------------------------------38
三、結果-----------------------------------------------------39
A.磷酸激酵素抑制劑對重金屬離子誘導MT基因轉錄的影響---39
抑制PKC活性--------------------------------------------------39
Ca/calmodulin抑制劑W7----------------------------------------40
磷酸水解酵素抑制劑okadaic acid和vanadate---------------------40
tyrosine kinase的抑制劑herbimycin A--------------------------41
MEK的抑制劑PD98059-------------------------------------------42
B.重金屬離子誘導金屬硫蛋白基因表現與細胞中Reactive oxygen--- species -----------------------------------------------------42
過氧化氫(H2O2)-----------------------------------------------42
Buthionine sulfoximine和N-acetyl-L-cysteine------------------43
3-amino-1,2,4-triazole---------------------------------------43
ascorbic acid和a-tocopherol----------------------------------44
Mannitol-----------------------------------------------------45
C. rottlerin對MT基因的調控-----------------------------------45
對PKC異構體有專一性的抑制劑對Cd或Zn誘發MT基因表達的影響------45
含MTF-1 cDNA片段表現載體的構築-------------------------------46
GST融合蛋白質的純化與分析------------------------------------46
Rottlerin能夠誘發CdR細胞中MT基因的表達-----------------------48
Rottlerin處理CdR細胞,不影響細胞中MTF-1的含量----------------49
Rottlerin促進MTF-1對MREs的結合活性---------------------------49
Rottlerin活化MTF-1的結合活性具專一性-------------------------50
Rottlerin抑制重金屬進入CdR細胞中-----------------------------51
Rottlerin誘導CdR細胞中MT蛋白質的增加-------------------------51
Rottlerin可以增加CdR細胞對Cd的抗性---------------------------52
細胞內的金屬螯合劑能夠抑制rottlerin引發的MTF-1的活化---------52
Rottlerin誘發MTF-1的活化及MT基因的表現具有細胞專一性---------53
四、討論-----------------------------------------------------54
五、引用文獻-------------------------------------------------64
六、附表-----------------------------------------------------83
七、附圖-----------------------------------------------------86
五、引用文獻
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