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研究生:黃爾毅
研究生(外文):Erh-Yi Huang
論文名稱:p21Waf1/CIP1/SDI1轉錄啟動子在人類髓幹細胞株D2細胞進行分化及凋零死亡兩種路徑中的調控機制之探討
論文名稱(外文):Regulation of the p21Waf1/CIP1/SDI1 promoter during TPA - induced differentiation and apoptosis in a myeloid D2 cell line
指導教授:張智芬
指導教授(外文):Zee-Fen Chang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:61
中文關鍵詞:p21Waf1/CIP1/SDI1轉錄啟動子分化凋零死亡
外文關鍵詞:p21Waf1/CIP1/SDI1 promoterdifferentiationapoptosis
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D2細胞是一種從人類髓幹細胞株TF-1衍生出來的GM-CSF independent的細胞株。經12-o-tetradecanoylphorbol-13-acetate (TPA)處理後,可造成進行分化 (differentiation)及凋零死亡(apoptosis)兩種形態的細胞。我們實驗室之前的研究發現,在TPA誘發分化的細胞中p21 mRNA的表現明顯上升,而在TPA誘發凋零死亡的D2細胞內,p21 mRNA未被誘發表現出來。
這個研究的目的,在於了解p21轉錄啟動子 (promoter) 在分化及凋零死亡兩種路徑中的調控機制。根據TPA誘導其他血液細胞走向分化的研究顯示,轉錄因子(transcription factor) Sp1及Ap2在活化p21轉錄啟動子的過程中扮演重要的角色。因此我們利用gel mobility shift的實驗,來探討轉錄因子Sp1和Ap2在TPA誘導D2細胞走向分化及凋零死亡兩種路徑中是否扮演重要的角色。結果顯示,轉錄因子Sp1的DNA結合活性在TPA誘導D2細胞走向分化及凋零死亡的過程中並沒有不同的變化。而Ap2的DNA結合活性在TPA處理後發生了明顯的改變。比較在走向分化及凋零死亡的D2細胞中,其Ap2-DNA結合活性的改變情形發現,雖然Ap2-DNA的結合模式(binding pattern)相同,但在Ap2-DNA complex A及 slowest Ap2-DNA mobility complex S的量上有明顯的差異。
另外,利用luciferase reporter 來研究p21的轉錄啟動子在TPA誘導D2細胞分化及凋零死亡兩種路徑中的表現。結果發現p21轉錄啟動子-209 ~ +11的區域即可受到TPA的調控。此外在p21轉錄啟動子-2326 ~ -209的區域似乎存在一個抑制的機制,而此抑制機制似乎在TPA調控p21轉錄啟動子上不扮演重要之角色。此抑制機制亦存在於另一株人類血球細胞株K562細胞中。
最後,我們還檢測不同類型的PKC在TPA誘導D2細胞走向分化及凋零死亡兩種路徑時的轉位情形。結果我們發現在走向分化及凋零死亡兩種路徑的D2細胞內,PKC α、β、γ、δ這四種類型的PKC在TPA誘導下都會轉移到細胞膜上並無明顯不同之處。
在本實驗中,我們發現在TPA誘導D2細胞走向分化及凋零死亡的兩種路徑中,轉錄因子Ap2的DNA結合形式有明顯的改變。此改變在走向兩種不同路徑的D2細胞中有量的變化。然而在走向分化及凋零死亡兩種不同路徑的D2細胞中, PKC被TPA活化的情形相似。因此在TPA誘導走向分化及凋零死亡的D2細胞中,其PKC的訊息傳遞路徑可能在PKC下游有所不同。因此造成Ap2的DNA結合形式有所不同。然而此不同是否和p21 mRNA在走向分化及凋零死亡的D2細胞中的表現情形有關必須透過進一步的實驗來證明。
D2, a variant cell line derived from the GM-CSF-dependent, human hematopoietic progenitor cells (TF-1), was established by culturing TF-1 in media lacking GM-CSF. With TPA treatment, there was about 54% of D2 cell attaching to the cultured flasks and undergoing differentiation. The cells remained in suspension underwent apoptosis. Expression of p21Waf1/CIP1/SDI1 mRNA was rapidly induced by TPA in the differentiating D2 cells, but not in the counter part of D2 cells undergoing apoptosis. The specific aim of this study is to understand why p21Waf1/CIP1/SDI1 is preferentially induced in the differentiating D2 cells.
Recent evidence showed that two transcription factors Sp1 and Ap2 are important for TPA-induced transcription of p21Waf1/CIP1/SDI1. Here, we found that the DNA binding activity of Sp1 is similar in differentiating and apoptotic D2 cells. As for Ap2, we found that TPA can induce a change in the DNA binding pattern of Ap2 in both differentiating and apoptotic D2 cells, there is a significant difference in the amount of Ap2-DNA complex A and slowest Ap2-DNA complex S between the differentiating and apoptotic D2 cells.
We further used the luciferase reporter system to study the regulation of the p21Waf1/CIP1/SDI1 promoter during TPA—induced differentiation and apoptosis in D2 cells. We found that the region from —209 to +11 of the p21Waf1/CIP1/SDI1 promoter can be up-regulated by TPA. Although a repression mechanisms mediated by —2322 to -209 of the p21Waf1/CIP1/SDI1 promoter exists in D2 and K562, it appeared that this repression mechanisms is not involved in TPA induced up-regulation of p21 expression.
Finally, we examined the membrane translocation of PKC in response to TPA treatment, we did not find difference in the activation of PKC isoforms and in the differentiating or apoptotic D2 cells.
Taken together, our data indicated that the difference in the Ap2-DNA complex A and slowest Ap2-DNA complex S found in nuclei extracts from differentiating and apoptotic D2 cells is correlated with the p21 promoter activity induced by TPA. Scince there was no difference in the translocation of some PKC isoforms in differentiating and apoptotic D2 cells, our results imply that the signal transduction pathway downstream of PKC activation might diverge in the TPA-induced ferentiating and apoptotic D2 cells. This divergence may be relevant to the slowest Ap2-DNA complex in TPA-induced differentiating and apoptotic D2 cells. However, it remians unclear whether the difference in the Ap2-DNA complex A and slowest Ap2-DNA complex S is involved in regulation of the p21Waf1/CIP1/SDI1 promoter activity.
中文摘要……………………………………………………………1
英文摘要……………………………………………………………3
緒論…………………………………………………………………5
材料與方法……………………………………………………… .12
實驗結果…………………………………………………………..25
討論………………………………………………………………..31
圖表………………………………………………………………..36
參考文獻………………………………………………………..…54
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