先前有許多文獻指出吸煙是引發肺癌的重要因子之ㄧ(1,2)。我們在先前(Toxicology and Applied Pharmacology, 2004)也發表過尼古丁引發致癌因子的是仰賴肺臟表皮細胞中尼古丁接受體將NF-Κb活化後會結合至cyclin D1的啟動子位置上，進而達到加速細胞生長且維持細胞存活的目的(3,4)。為了深入探討吸煙習慣和乳癌發生率的相關性(5-7)，因此我們認為在乳癌的發生部位應該可以找到特定尼古丁接受體(nAchR)。在本實驗中我們在臺灣女性乳癌病人的腫瘤中找到特定的尼古丁接受體(nAchR)，我們也是第一個發現在乳癌細胞株MCF-7和MDA-MB-231有共同的α5、α9尼古丁接受體。接著我們收集了四十位女性乳癌病人的正常及腫瘤部位做個別尼古丁在mRNA上的表現中發現nAchR ubtype α9及α10是在正常及腫瘤組織中最常發現的尼古丁接受體。利用即時性PCR (RealTime-PCR)定量mRNA的表現也發現腫瘤組織中α9尼古丁接受體的表現量有明顯的高於正常組織中α9尼古丁接受體的表現量的情況。接著在西方墨點法中也發現經由尼古丁刺激細胞而調控的蛋白最主要是依靠PI3K/AKT訊息傳遞路徑。我們也利用了Si RNA的技術做出了抑制α5和/或α9尼古丁接受體蛋白表現的MDA-MB-231乳癌細胞株，結果發現抑制α9尼古丁接受體的細胞生長速率明顯的比未抑制α9尼古丁接受體的乳癌細胞慢，以上的結果可能顯示α9尼古丁接受體可能在尼古丁所引發的乳癌中扮演非常重要的角色。

Previous study indicated that tobacco-smoking is a well nderstanding carcinogenic factors that promote the lung cancer formation(1,2). The mechanisms of nicotine-induced carcinogenesis were demonstrated in our recent report (Toxicology and Applied Pharmacology, 2004, in press) indicated as specific binding of nicotine to the nicotinic acetylcholine receptor (nAchRs) in lung epithelial cells by activation of the cyclin D1 promoter through the NFkB signaling proteins to induced cell proliferation(3,4). To further investigate the tobacco smoking habits and its correlations of breast tumor formation(5-7), therefore, we suggested that specific nAchR subunits will be identified in the breast tumor. In this study, our results demonstrated that (nAchR) was detected in breast tumor tissue in Taiwanese patients. We first demonstrated that the a5 and a9 nAchRs were detected in both the MCF-7 and MDA-MB-231 breast cancer cell lines. We further study the expression of the nAchR mRNA levels in breast tumor tissues collected from forty cases of breast cancer patients in Taiwan and found that the α9 and α10 subunits of the nAchR was the most prevalence in both normal and tumor tissue. Quantitative assays of the mRNA levels of the nAchRs was measured by Real-time PCR analysis technique and revealed that expression of the a9-nAchR was higher in the tumor tissue when compared to the normal tissue which dissected form the tumor margin. Western blotting analysis was then performed and demonstrated that the PI3K/Akt-regulated proteins were the major signaling pathway that involved in cell proliferation stimulated by nicotine treatment. Different clones of cell lines that inhibited the a9 anda5 receptor expression by SiRNA technique was established in the MDAMB 231 cells. We found that cell growth curve was significant inhibited in the a9-siRAN-nAchR cells. Such results implied that the a9-nAchR may play some important role involved in nicotine-mediated breast tumor carcinogenesis.

目錄
中文摘要
英文摘要
第一章 緒論
一、AKT路徑
二、吸煙和乳癌

第二章 實驗材料與方法
一、實驗材料
（一）、試劑與藥品
（二）、常用儀器
(三) 、常用溶液
二、實驗方法
(一)、細胞培養
(二)、 細胞生長曲線之測定
(三)、反轉錄–聚合酶連鎖反應
(四)、西方墨點法


第三章 實驗結果
ㄧ、人類乳癌細胞株的nAchR表現型態
二、人類乳癌細胞株對於尼古丁的訊息傳遞路徑
三、利用抑制劑確認尼古丁的活化路徑
四、台灣地區乳癌細胞的nAchR表現型態
五、即時性定量比較nAchRα9在正常及腫瘤組織的表現
六、利用siRNA抑制nAchRα9並觀察乳癌細胞的生長狀況

第四章、討論

第五章、圖表
Figure 1a, 1b
Figure 2
Figure 3
Figure 4
Table 1a
Table 1b
Figure 5a, 5b
Figure 5c
Figure 5d
Figure 5e
Figure 6
Figure 7
Figure 8

第五章、參考文獻

1.Proctor, R. N. (2001) Nat Rev Cancer 1(1), 82-86
2.Shields, P. G. (2002) Oncogene 21(45), 6870-6876
3.Anto, R. J., Mukhopadhyay, A., Shishodia, S., Gairola, C. G., and Aggarwal, B. B. (2002) Carcinogenesis 23(9), 1511-1518
4.West, K. A., Brognard, J., Clark, A. S., Linnoila, I. R., Yang, X., Swain, S. M., Harris, C., Belinsky, S., and Dennis, P. A. (2003) J Clin Invest 111(1), 81-90
5.Schumacher, H. H. (2005) Ann Plast Surg 54(2), 117-119
6.Reynolds, P., Hurley, S., Goldberg, D. E., Anton-Culver, H., Bernstein, L., Deapen, D., Horn-Ross, P. L., Peel, D., Pinder, R., Ross, R. K., West, D., Wright, W. E., and Ziogas, A. (2004) J Natl Cancer Inst 96(1), 29-37
7.Gammon, M. D., Schoenberg, J. B., Teitelbaum, S. L., Brinton, L. A., Potischman, N., Swanson, C. A., Brogan, D. J., Coates, R. J., Malone, K. E., and Stanford, J. L. (1998) Cancer Causes Control 9(6), 583-590
8.Brognard, J., Clark, A. S., Ni, Y., and Dennis, P. A. (2001) Cancer Res 61(10), 3986-3997
9.West, K. A., Linnoila, I. R., Belinsky, S. A., Harris, C. C., and Dennis, P. A. (2004) Cancer Res 64(2), 446-451
10.Giovino, G. A. (2002) Oncogene 21(48), 7326-7340
11.Hecht, S. S. (2003) Nat Rev Cancer 3(10), 733-744
12.Dajas-Bailador, F. A., Soliakov, L., and Wonnacott, S. (2002) J Neurochem 80(3), 520-530
13.Minna, J. D. (2003) J Clin Invest 111(1), 31-33
14.Nakayama, H., Numakawa, T., Ikeuchi, T., and Hatanaka, H. (2001) J Neurochem 79(3), 489-498
15.Yildiz, D. (2004) Toxicon 43(6), 619-632
16.Nakayama, H., Numakawa, T., and Ikeuchi, T. (2002) J Neurochem 83(6), 1372-1379
17.Evan, G. I., and Vousden, K. H. (2001) Nature 411(6835), 342-348
18.Cantley, L. C. (2002) Science 296(5573), 1655-1657
19.Brazil, D. P., Park, J., and Hemmings, B. A. (2002) Cell 111(3), 293-303
20.Aoki, M., Blazek, E., and Vogt, P. K. (2001) Proc Natl Acad Sci U S A 98(1), 136-141
21.Datta, S. R., Brunet, A., and Greenberg, M. E. (1999) Genes Dev 13(22), 2905-2927
22.Nicholson, K. M., and Anderson, N. G. (2002) Cell Signal 14(5), 381-395
23.Vivanco, I., and Sawyers, C. L. (2002) Nat Rev Cancer 2(7), 489-501
24.Franke, T. F., Kaplan, D. R., and Cantley, L. C. (1997) Cell 88(4), 435-437
25.Al-Delaimy, W. K., Cho, E., Chen, W. Y., Colditz, G., and Willet, W. C. (2004) Cancer Epidemiol Biomarkers Prev 13(3), 398-404
26.Innes, K. E., and Byers, T. E. (2001) Cancer Causes Control 12(2), 179-185
27.Morabia, A. (2002) Environ Mol Mutagen 39(2-3), 89-95
28.Murin, S., Pinkerton, K. E., Hubbard, N. E., and Erickson, K. (2004) Chest 125(4), 1467-1471
29.Shrubsole, M. J., Gao, Y. T., Dai, Q., Shu, X. O., Ruan, Z. X., Jin, F., and Zheng, W. (2004) Int J Cancer 110(4), 605-609
30.Mei, J., Hu, H., McEntee, M., Plummer, H., 3rd, Song, P., and Wang, H. C. (2003) Breast Cancer Res Treat 79(1), 95-105
31.Narayan, S., Jaiswal, A. S., Kang, D., Srivastava, P., Das, G. M., and Gairola, C. G. (2004) Oncogene 23(35), 5880-5889
32.Cormier, A., Paas, Y., Zini, R., Tillement, J. P., Lagrue, G., Changeux, J. P., and Grailhe, R. (2004) Mol Pharmacol 66(6), 1712-1718
33.Conroy, W. G., and Berg, D. K. (1995) J Biol Chem 270(9), 4424-4431
34.Karlin, A. (2002) Nat Rev Neurosci 3(2), 102-114
35.Nordberg, A. (2001) Biol Psychiatry 49(3), 200-210
36.Song, P., Sekhon, H. S., Jia, Y., Keller, J. A., Blusztajn, J. K., Mark, G. P., and Spindel, E. R. (2003) Cancer Res 63(1), 214-221
37.Cooper, S. T., and Millar, N. S. (1998) J Neurochem 70(6), 2585-2593
38.Vailati, S., Moretti, M., Longhi, R., Rovati, G. E., Clementi, F., and Gotti, C. (2003) Mol Pharmacol 63(6), 1329-1337
39.Brown, E. N., and Galligan, J. J. (2003) Am J Physiol Gastrointest Liver Physiol 285(1), G37-44
40.Lindstrom, J. M. (2003) Ann N Y Acad Sci 998, 41-52
41.Loden, M., Stighall, M., Nielsen, N. H., Roos, G., Emdin, S. O., Ostlund, H., and Landberg, G. (2002) Oncogene 21(30), 4680-4690
42.Elwood, J. M., and Burton, R. C. (2004) Med J Aust 181(5), 236-237
43.Manjer, J., Johansson, R., and Lenner, P. (2004) Int J Cancer 112(2), 324-328
44.Dunn, B. K., Wickerham, D. L., and Ford, L. G. (2005) J Clin Oncol 23(2), 357-367
45.Catalano, M. G., Frairia, R., Boccuzzi, G., and Fortunati, N. (2005) Mol Cell Endocrinol 230(1-2), 31-37
46.Kushner, P. J., Agard, D. A., Greene, G. L., Scanlan, T. S., Shiau, A. K., Uht, R. M., and Webb, P. (2000) J Steroid Biochem Mol Biol 74(5), 311-317
47.Stoica, G. E., Franke, T. F., Wellstein, A., Czubayko, F., List, H. J., Reiter, R., Morgan, E., Martin, M. B., and Stoica, A. (2003) Mol Endocrinol 17(5), 818-830