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博碩士論文 etd-0810112-224956 詳細資訊
Title page for etd-0810112-224956
論文名稱
Title
基因放大驅使BCL6過度表現位於膀胱的尿路上皮癌
Amplification-driven BCL6 overexpression in urothelial carcinoma of urinary bladder
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
42
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2012-07-16
繳交日期
Date of Submission
2012-08-10
關鍵字
Keywords
膀胱癌、晶片式全基因體定量分析技術、致癌基因、細胞增值、細胞轉移
BCL6, aCGH, oncogene, proliferation, migration
統計
Statistics
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中文摘要
膀胱的尿路上皮癌是泌尿道最常見的腫瘤。大約70%的腫瘤診斷屬於非侵襲
性的腫瘤。非侵襲性的腫瘤通常長在膀胱多處的地方,利用外科手術去除或是化
學療法治療,但這些腫瘤可能會再次復發影響病患。然而這些非侵襲性的腫瘤很
少侵犯到肌肉的階段且5年的存活率接近90%。其餘的30%的腫瘤診斷屬於侵襲性
的腫瘤,儘管膀胱切除、化學治療或是放射線治療,大於50%的侵襲性腫瘤會轉
移到其的它器官,該轉移行為截至目前仍缺乏良好預測因子,且一旦發生轉移,5
年存活率大約僅剩6%。因此,進一步尋找與膀胱癌侵犯行為相關分子標的以協助
臨床預後評估及開發新的治療策略是膀胱癌研究上的重大課題。BCL6是前致癌基
因的編碼為細胞核轉錄的抑制者,它會抑制DNA的修補和TP53的活性。這兩種原
因的影響之下,導致正常細胞對DNA修補去功能增加個體的突變,在腫瘤中,BCL6
的突變可能促進腫瘤的惡化。一些研究已經證明BCL6在不同形式的腫瘤所扮演的
角色,然而BCL6在膀胱癌的作用機制目前還不知道。因此,本實驗利用膀胱癌的
細胞株包括了BFTC905和J82,探討內生性的BCL6 mRNA和蛋白質表現量。接著利
用shRNA干擾技術抑制BCL6基因的表現並且分析BCL6對細胞的癌化程度的關
係。接著分析它對細胞的存活率、轉移能力和侵襲能力的影響。相對的,也讓膀
胱癌細胞株過度表現BCL6然後評估細胞的增值、轉移能力和侵襲能力。最後進行
體內實驗來研究BCL6在生物體的影響。藉由本實驗,我們將進一步釐清BCL6與膀
胱癌侵犯性的關連以及釐清BCL6是否適合被視為膀胱癌的分子治療標的。
Abstract
Urinary bladder urothelial carcinoma is the most common cancer of the urinary
tract. About 70% of the diagnosed tumors classified as Non-invasive tumor, which is
usually multiple. Despite surgical removal and perioperative chemotherapy, tumor
recurrence is not uncommon. However, the chance for such non-invasive tumors to
advance to the muscle-invasive stage is relatively small and the 5-year survival rate
approaches 95%. The rest 30% are classified as invasive tumors which usually pursue
aggressive clinical course. In spite of radical cystectomy in conjunction with
debilitating chemotherapy and/or radiotherapy, more than 50% of invasive tumors
eventually spread to distant organs. The 5-year survival rate for patients with distant
metastasis is only about 6%. The current challenge in the management of urinary
bladder carcinoma is the lack of powerful prognostic marker and promising therapeutic
agents. Accordingly, to identify novel biomarks to adjust therapeutic strategy is
mandatory. The BCL6 proto-oncogene encodes a nuclear transcriptional repressor, it’s
inhibits DNA repair pathways and TP53. Perturbation of both these pathways may
contribute to normal cell function by repressing DNA damage responses and permitting
somatic hypermutation but , in the context of malignancy, this could lead to mutations
promoting aggressive tumor. Several studies have demonstrated that BCL6 play a role in
different cancer types, however, the function of BCL6 in bladder cancer is understood.
Therefore, in this study, we will analyze the endogenous BCL6 mRNA and
total/activated BCL6 protein in various bladder cancer cell lines, including BFTC905,
and J82. Then we will knockdown of the BCL6 gene by shRNA interference and
analyze how it implicates various cellular processes essential to cancerous states. And
then we will be analyzed the affection of cell survival, migration and invasion.
Conversely, Overexpression of BCL6 in bladder cancer cell lines will be assessed cell
proliferation, migration and invasion. Finally, studying it’s affection in vivo. We
demonstrate that BCL6 is correlated with bladder cancer.
目次 Table of Contents
目錄
中文摘要 .................................................................................................................................. i
Abstract................................................................................................................................... ii
一、緒論 ................................................................................................................................. 3
1.膀胱癌 ............................................................................................................................... 3
(1)膀胱癌的危險因子 ...................................................................................................... 3
(2)膀胱癌的病理症狀 ...................................................................................................... 4
(3)膀胱癌的分期和治療 .................................................................................................. 4
(4)膀胱癌和基因的關係 .................................................................................................. 4
2.BCL6 基因和BCL62 蛋白質 ........................................................................................... 5
二、材料與方法 ...................................................................................................................... 6
1.細胞培養 ........................................................................................................................... 6
2.RNA 萃取 .......................................................................................................................... 6
3.Quantitative Real-Time PCR ............................................................................................. 7
4.免疫組織化學染色法 ....................................................................................................... 7
5.西方墨點法 ....................................................................................................................... 7
6.RNAi ................................................................................................................................ 8
7.BrdU assay......................................................................................................................... 9
8.細胞週期分析 ................................................................................................................... 9
9.Wound healing assay ......................................................................................................... 9
10.Cell invasion assay ........................................................................................................ 10
11.統計分析 ....................................................................................................................... 10
三、結果 ............................................................................................................................... 11
1.利用Array comparative genomic hybridization 分析膀胱癌病人中,染色體增幅的
情況 ................................................................................................................................... 11
2
2.比較膀胱癌病人中,在不同tumor stage 中BCL6 mRNA 和BCL6 protein 的表現
量 ....................................................................................................................................... 11
3.BCL6 表現量的高低,對於膀胱癌患者存活率的影響 ............................................... 12
4.確認膀胱癌細胞株中BCL6 的表現量 ......................................................................... 12
5.確認BFTC905 細胞株中BCL6 的表現量是否被抑制 ................................................ 12
6.確認J82 細胞株中BCL6 的表現量是否有上升 .......................................................... 13
7.BCL6 基因過度表現或表現量受抑制時,對於膀胱癌細胞株proliferation 能力的影
響 ....................................................................................................................................... 13
8.BCL6 基因過度表現或表現量受抑制時,對膀胱癌細胞株中cell cycle 的影響 ...... 14
9.BCL6 基因過度表現或表現量受抑制時,對於膀胱癌細胞株中,migration 能力的
影響 ................................................................................................................................... 14
10.BCL6 基因過度表現或表現量受抑制時,對於膀胱癌細胞株中,invasion 能力的
影響 ................................................................................................................................... 15
四、圖表 ............................................................................................................................... 16
五、討論 ............................................................................................................................... 28
六、參考文獻 ........................................................................................................................ 31
七、附錄 ............................................................................................................................... 36
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