臺灣博碩士論文加值系統

中文摘要
本篇研究計畫主要在探討中草藥厚朴的酚類萃取物質，magnolol ，對於促進p21蛋白質在大腸癌細胞(colo205)內部表現量的分子機轉。本實驗室先前發現magnolol可以藉由增加細胞內部p21蛋白質的表現量而抑制大腸癌細胞(colo205)以及肝癌細胞(Hep-G2與Hep-3B)的生長。這些p21蛋白質可與細胞週期蛋白酵素CDK2結合，而抑制CDK2的活性，進而導致細胞停滯在G0/G1 phase。然而，針對magnolol調控p21 蛋白上升的上游訊號傳遞機制，目前不十分的清楚。因此本篇研究的目的是利用大腸癌細胞(colo205)為材料來研究magnolol促進p21 蛋白表現增加的分子機轉。本實驗室之前證實低濃度的處理條件之下，magnolol確實會抑制colo205細胞的生長。此外，我們藉由Western blot分析magnolol之處理在一個小時內對於colo205細胞的訊號傳遞蛋白分子之間的影響。結果發現magnolol處理大腸癌細胞一個小時內會啟動Ras/Raf/Erk的訊號傳遞機制；利用Ras抑制劑預先處理大腸癌細胞，我們發現magnolol誘發細胞一個小時內會活化的Erk則有被抑制的現象，並且在magnolol處理18小時時所調控上升的p21蛋白質表現量也被抑制；此時， magnolol所調控的細胞週期停滯現象也消失。而在處理大腸癌細胞的24小時內觀察p21的mRNA的變化，結果發現在第一個小時p21的mRNA即大量轉錄表現。由於之前的研究顯示Staurosporine能阻止magnolol調控p21蛋白質表現上升現象，因此我們觀察二十四小時內以及一小時內colo205細胞內的PKC是否有活化的發生。結果發現magnolol處理大腸癌細胞colo205之後的一個小時內， PKC沒有產生活化的現象。然而，在18小時時PKC產生活化的現象(即membrane translocation)。此外， Staurosporine也會抑制一個小時內的Erk活化現象。因為細胞週期的調控途徑非常複雜，包括Ras/Raf, PKA, PI3K等，由於PI3K抑制劑(wortmannin)與PKA抑制劑(H89)皆不會影響magnolol所調控的細胞週期停滯現象。因此，我們推測magnolol調控細胞內部的p21蛋白質表現量的上升主要是藉由Ras/Raf-1/Erk的訊號傳遞機制來提高p21的蛋白質表現量，然而此結果不排除尚有其他的訊號傳遞機制也會受magnolol的調控而導致p21的表現量上升，以及細胞週期停滯。

Abstract
In Taiwan, the most populations of mortality are caused by cancer, including colon carcinoma. Previously, we have demonstrated that magnolol inhibited cultured human colon cancer cell growth by up-regulation of p21 protein. The molecular mechanism underlying of magnolol-induced up-regulation of p21, however, is still unclear. Previous study showed that a PKC inhibitor, staurosporine, abolished the magnolol-induced p21 up-regulation. PKC, a family of serine-threonine kinase, has been implicated in the regulation of colon tumorgenesis. Here we demonstrated that translocation of PKC γ was observed at 18 hour after treatment of colon cancer cell line colo205 with magnolol, and the magnolol-induced PKC γ translocation was prevented by PD98059, an Erk1/2 antagonist. PD98059 treatment also blocked the magnolol-induced p21 up-regulation in colo205 cells, suggesting that Erk1/2 might be involved in magnolol-induced p21 up-regulation activation of PKC γ. Recent report indicated that activation of Ras gene can increase p21 protein expression via Ras/Raf/Erk signal pathway. Accordingly, we hypothesize that magnolol-induced p21 elevation via Ras/Raf/Erk signal pathway. Treatment of colo205 cells by magnolol increased Erk1/2 phosphorylation. Moreover, pretreatment of the cell with magnolol-induced elevation of p21 and cell cycle arrest were inhibited by Ras inhibitor peptide (VPPPVPPRRR). An increase of Raf-1 (ser338) phosphorylation was also observed after magnolol treatment in colo205 cells. Taken together, our data suggest that magnolol-induced colo205 cell cycle arrest and up-regulation of p21 might be mediated through Ras/Raf/Erk signal pathway.

目次
中文摘要 …………………………………………………………… 1
英文摘要 …………………………………………………………… 3
圖目錄 ……………………………………………………………… 6
表目錄 ……………………………………………………………… 7
緒論 ………………………………………………………………… 8
一, 大腸癌 …………………………………………………… 8
二, 細胞週期 ………………………………………………… 10
三, Ras family對細胞的影響 ……………………………….. 13
研究方向與目的 ……………………………………………………17
研究方法與材料 ……………………………………………………19
一、 細胞培養 ……………………………………………… 19
二、 西方墨點法 …………………………………………… 21
三、 H3 thymidine incorporation ……………………………30
四、 厚朴 ………………………………………………………32
五、 常用緩衝溶液及試劑 ……………………………………35
實驗結果分析 ……………………………………………………… 38
討論 ………………………………………………………………… 49
參考文獻 …………………………………………………………… 53
附圖及圖 …………………………………………………………… 59
附圖目錄
附圖一 : Magnolol的結構式 ……………………………………….. 59
附圖二 : 大腸癌的發展過程 ………………………………………. 60
附圖三 : 細胞週期進行的過程 ……………………………………..61
附圖四 : Ras Protein cycle …………………………………………… 62
附圖五 : Ras family signal pathway ………………………………… 63
附圖六 : 總結 ………………………………………………………. 64
圖目錄
Fig-1 Magnolol 處理colo 205細胞株，觀察PKC的活化現象 ...…65
Fig-2 magnolol調控colo205細胞24小時內p21的表現 …………...67
Fig-3 Magnolol 50μM對於colo205細胞一個小時內PKC的影響 …68
Fig-4 PKC抑制劑RO對Magnolol所引發的細胞週期停滯的影響…69
Fig-5 Staurosporine 的預先處理之下對於Magnolol調控colo205細胞的影響…………………………………………………………………..…70
Fig-6 magnolol對於colo205細胞Raf-1的調控……………………..71
Fig-7 magnolol對於colo205細胞Erk1/2的調控……………………72
Fig-8 Magnolol對於colo205細胞一個小時內的Ras的影響……......73
Fig-9 Ras抑制劑處理之下，magnolol對colo205細胞內Erk的影響74
Fig-10 Ras inhibitor peptide抑制magnolol所調控上升的p21蛋白質……………………………………………………………………... 75
Fig-11 U73122對magnolol調控colo205細胞內Erk activity的影響……….……………………………………………………………...76
Fig-12 PI3K , PKA以及CaM kinase的抑制劑對magnolol所調控的細胞週期停滯的影響……………………………………………………78

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