膀胱癌 (Freedman et al.)，為一種從尿路上皮 (Pich et al.) 衍生而來的癌症。為國民十大癌症之一，並具有高死亡率與高醫療成本。在過去研究的資料中發現當交集BCL6高度表現時，會促進膀胱癌細胞週期分布與增生，為一著名之致癌基因。在本篇研究將探討BCL6透過何種路徑影響細胞週期分布與增生，本實驗室過去利用全基因表現矩陣分析 (HumanHT-12v4 Expression BeadChip)中分析，當BCL6上升與靜默有顯著上升下降的前2900個基因並進一步交集得到1310個基因，利用KEGG mapper分析，發現細胞週期路徑與Forkhead box protein O (FOXO)路徑。BCL6透過FOXO pathway，進而影響FOXO3 (Forkhead box O3)之表現。為了證明BCL6透過何種路徑影響FOXO3之表現量與其核質分布，進而影響細胞週期與增生。FOXO3透過其磷酸化位點來調節其功能與表現量，因此利用上調BCL6基因，並且添加LY294002 (PI3K inhibitor)、AZD6244 (MEK/ERK inhibitor)與Gefitinib (EGFR inhinitor)觀察FOXO3磷酸化位點變化、核質分布與細胞週期，發現T24與BFTC905膀胱癌細胞之BCL6透過MEK/ERK，影響下游基因與細胞功能之表現。因此得出結論調節BCL6之活性是一種具有潛在性之治療方式。

Bladder cancer is derived from urothelial, called urothelial carcinoma. It’s the most top ten cancer in Taiwan. In addition, the cancer with high mortality rate and cost of treatment. In past report, the BCL6 induced cell cycle progression and cell proliferation when the BCL6 highly expression in bladder cancer. According to the analysis of whole gene array (HumanHT-12v4 Expression BeadChip) find that 2900 genes regulated by the BCL6. Further analysis of intersection gets 1310 genes. Using the KEGG mapper analysis finds that cell cycle pathway and the FOXO pathway regulated by the BCL6. The BCL6 through the FOXO pathway effects the FOXO3 (Forkhead box O3) expression. To prove the BCL6 how to effect the FOXO3 expression level and nuclear/cytosol distribution, and then affect the cell cycle progression and cell proliferation. The FOXO3 regulates cell function and expression level through the phosphorylation site. For the reason that, to up regulates the BCL6 and add to LY294002 (PI3K inhibitor), AZD6244 (MEK inhibitor), Gefitinib (EGFR inhibitor) to observed the change of phosphorylation site and nuclear/cytosol distribution and cell cycle progression of the FOXO3. Understanding BCL6 effects the downstream genes and cell function through the MEK/ERK in T24 and BFTC905 bladder cancer cell lines. Therefore, to control the activity of BCL6 is a potential therapy.

論文審定書 i
致謝 ii
摘要 iii
Abstract iv
目錄 v
圖次 vii
表次 viii
1、 緒論 1
1.1、簡介膀胱癌 1
1.2、簡介BCL6 2
1.3、簡介FOXO 3
1.4、簡介PI3K pathway 4
1.5、簡介MAPK pathway 4
2、實驗材料與方法 5
2.1、 細胞 5
2.1.1、細胞培養 5
2.1.2、 細胞解凍 5
2.1.3、 繼代培養 6
2.1.4、 細胞冷凍保存 6
2.2、質體製備 7
2.2.1、 質體 7
2.2.2、 質體轉型 7
2.2.3、 質體純化 7
2.2、 基因轉染(transfection) 8
2.3.1、Transient transfection 8
2.3.2、慢病毒感染 (Lentiviral infection) 9
2.4、 cDNA製備與Quantitative real-time PCR (QPCR) 9
2.4.1、 Total RNA萃取 9
2.4.2、 反轉錄聚合酶連鎖反應 (Reverse transcription) 10
2.4.3、 Quantitative real-time (qPCR) 10
2.4.4、 瓊脂膠體電泳 (Agarose gel electrophoresis) 10
2.5、 蛋白質分析 11
2.5.1、蛋白質純化 11
2.5.2、 蛋白質定量 11
2.5.3、 Acrylamide gel 製作 11
2.5.4、 西方點墨法 (western blotting) 12
2.6、核質分離 13
2.7、細胞免疫化學染色 (Immunocytochemistry) 13
3、 結果 21
3.1、BCL6過度表現，影響下游mRNA與蛋白質之表現量 21
3.2、 BCL6基因靜默，影響下游mRNA與蛋白質之表現量 21
3.3、 BCL6 透過MAPK路徑影響細胞週期分布與CDKN1A、CDKN1B之蛋白質表現量 24
3.4、 BCL6透過PI3K路徑影響細胞週期分布與CKKN1A、CKKN1B蛋白質表現量 25
3.5、 BCL6透過EGFR影響細胞週期分布與CDKN1A、CDKN1B之表現量 25
3.6、 在膀胱癌細胞株T24、BFTC905，分別轉染BCL6質體，與基因靜默BCL6觀察FOXO3在細胞核與細胞質之分布。 30
4、討論 33
5、參考文獻 34

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