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研究生:張雅捷
研究生(外文):Ya-Chieh Chang
論文名稱:雌二醇與尼古丁透過PI3K/Akt和MAPK訊息傳遞路徑促進MCF-7人類乳腺癌細胞中尼古丁受器nAChRalpha9基因表現的機制探討
論文名稱(外文):17 beta-estradiol (E2) and Nicotine Can Increase Nicotinic Acetylcholine Receptor alpha9 ( nAChR alpha9 ) Gene Expression Through PI3K/Akt and MAPK Signaling Transduction Pathway in MCF-7 Human Breast Cancer Cells
指導教授:何元順
指導教授(外文):Yuan-Soon Ho
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗生物技術學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:76
中文關鍵詞:雌二醇尼古丁PI3K/Akt路徑MAPK路徑MCF-7人類乳腺癌細胞尼古丁受器nAChR alpha9
外文關鍵詞:17 beta-estradiol (E2)nicotinenicotinic acetylcholine receptor alpha9 (nAChR alpha9)PI3K/AktMAPKMCF-7 human breast cancer
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本研究主要目的是探討乳癌、雌性素、尼古丁三者的關聯性。欲知雌二醇和尼古丁如何調控MCF-7乳腺癌細胞株中尼古丁受器nAChR alpha9的基因表現與兩者在細胞內共同的訊息傳遞路徑,並且尋找主要影響尼古丁受器基因轉錄的轉錄因子。當MCF-7細胞株給予生理濃度的雌二醇 ( 1 nM ) 和尼古丁 ( 10 microM ) 時,其尼古丁受器的mRNA可在48小時後明顯增加。而在細胞訊息傳遞方面:使用PI3K抑制劑LY 294002,以及MAPK抑制劑PD 98059和SP 600125,可抑制住兩者共同的訊息傳遞路徑。因此歸納出兩者共同訊息傳遞路徑為PI3K/ Akt/p-ER alpha( Ser 167 ) 以及MEK1,2/ ERK1,2/ JNK1,2/p-ER alpha ( Ser 118 ) 。此外,運用Serial deletion luciferase activity assay和Site-directed mutagenesis assay發現雌二醇影響尼古丁受器基因轉錄的轉錄因子結合位置為VDR和AP-1,而尼古丁影響尼古丁受器基因轉錄的轉錄因子結合位置為AP-1。
The purpose of this investigation is to study the effects of 17 beta-estradiol and nicotine on nicotinic acetylcholine receptor alpha9 ( nAChR alpha9 ) gene expression and the cross-linking signaling pathways in MCF-7 human breast cancer cells. Using physiological concentrations of 17 beta-estradiol ( 1 nM ) and nicotine ( 10 microM ) could increase nAChR alpha9 mRNA expression after 48 hours treatment. Using PI3K inhibitor, LY 294002, could block both cross-linking pathway in MCF-7. PD 98059 and SP 600125, inhibitors of mitogen-activated protein kinase (MAPK) pathways, were also involved in both cross-linking pathways in MCF-7. Using serial deletion luciferase activity assay and site-directed mutagenesis assay to predict the most important transcription factor binding sites on nAChR alpha9 gene promoter. These results demonstrate the cross-linking pathways of 1 nM 17 beta-estradiol and 10 microM nicotine were through PI3K/Akt/p-ER alpha( Ser 167 ) and MEK1, 2/ ERK1,2/JNK1,2/p-ER alpha( Ser 118 ). The most important transcription factor binding sites on nAChR alpha9 gene promoter for 17 beta-estradiol are Vitamin D Receptor ( VDR ) and Activated Protein- 1 ( AP-1 ). The most one for nicotine is Activated Protein- 1 ( AP-1 ).
中文摘要 ( Abstract in Chinese ) I
英文摘要 ( Abstract in English ) III
目 錄 ( Contents ) V
圖 目 錄 ( Figures and Tables lists ) VIII
縮寫表 ( Abbreviations ) X

第一章 緒論 ( Introduction )
前言 2
一、乳癌與荷爾蒙的關係 3
二、Estrogen receptor ( ER ) 的介紹 4
三、Nicotinic acetylcholine receptors ( nAChRs ) 的介紹 5
四、Phosphoinositide 3-kinase ( PI3K ) / Akt signaling pathway 的介紹 6
五、Mitogen-activated protein kinase ( MAPK ) signaling pathway 的介紹 7

第二章 實驗材料與方法 ( Materials and Methods )
(一)藥品試劑 10
(二)常用溶液 14
(三)常用儀器 17
(四)實驗方法
1.細胞培養 Cell Culture 18
2.反轉錄-聚合酶連鎖反應 RT-PCR 19
3.西方墨點法 Western Blotting Assay 21
4.連續刪除片段生物冷光活性分析Serial Deletion Luciferase Activity Assay 22
5.核質分離 Nucleus Extraction 25
6.染色質免疫沉澱法 Chromatin Immunoprecipitation;ChIP Assay 27
7.點突變試驗 Site-directed Mutagenesis Assay 29

第三章 實驗結果 ( Results )
一、Nicotine 在 MCF-7 乳腺癌細胞中能引起反應的最佳劑量為 10 microM 33
二、17 beta-estradiol 在 MCF-7 乳腺癌細胞中能引起反應的最佳劑量為 1 nM 34
三、1 nM 17 beta-estradiol 和 10 microM nicotine 會使 MCF-7 乳腺癌細胞的 nAChR alpha9 在 48 小時表現最明顯 35
四、單獨或合併給予 1 nM 17 beta-estradiol 和 10 microM nicotine 作用於 MCF-7 乳腺癌細胞株的訊息傳遞路徑蛋白表現情形 36
五、17 beta-estradiol 和 nicotine 在 MCF-7 乳腺癌細胞中 PI3K/Akt 和 MAPK 訊息傳遞路徑 36
六、利用 Serial deletion luciferase activity assay 和 Site-directed mutagenesis assay 預測出 17 beta-estradiol 主要的轉錄因子結合位置在 VDR 和 AP-1 而 nicotine 主要的轉錄因子結合位置 AP-1 38
七、利用 nAChR alpha9 siRNA 細胞株與 MCF-7 細胞株做比較 39

第四章 討論 ( Discussion ) 41
第五章 圖表 (Figures ) 45
第六章 文獻參考 (References ) 66

附錄 ( Appendices )
一.pGL3 vector map and nAChR alpha9 vector map 73
二.The chemical structures of 17 beta-estradiol and nicotine 74
三.人體雌性素含量 75
四.本篇論文結論圖 76
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