博碩士論文 953403049 詳細資訊




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姓名 陳紹寬(Shao-Kuan Chen)  查詢紙本館藏   畢業系所 機械工程學系
論文名稱 靜水壓力增強絲裂霉素C對泌尿上皮癌 感受性的基因途徑
(Genetic pathway in urothelial carcinoma by Hydrostatic pressure synchronize with Mitomycin C)
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摘要(中) 摘要
泌尿上皮細胞癌 (urothelial carcinoma) 是泌尿生殖系統中第二常見的癌症(第一是攝護腺癌),是膀胱癌的最主要成分。臨床上對於表淺性膀胱癌(沒有侵犯肌肉層)標準的治療為經尿道內視鏡腫瘤切除術 (TURBT),通常在經尿道內視鏡腫瘤切除術後加以膀胱內免疫藥物灌注治療或化學藥物灌注治療以降低其復發率。化學藥物灌注比起免疫藥物灌注有比較少的副作用,但對於復發率降低則有比較少的效果。在 TURBT 後加以絲裂霉素C (MMC ) 膀胱化學藥物灌注治療是目前標準治療的策略以預防其術後復發,因此如何增加 MMC 化學藥物膀胱灌注治療功效,具有臨床醫療的重要性。
本文以膀胱癌細胞株 BFTC 905 cells 進行靜水壓 (Hydrostatic pressure) 合併 MMC 化學藥物膀胱灌注治療功效的研究,膀胱癌細胞生存能力以細胞存活率分析法 (MTT) 來評估。以流式細胞儀 (flow cytometry) 來評估細胞凋亡(apoptosis),細胞凋亡功能的改變則以分析細胞凋亡蛋白酶 caspase 3/7 的活力, Fas配體(FasL) 的表現力,以及粒線體膜電位損失 (loss of mitochondria membrane potential) 來評估。結果顯示,靜水壓的增加可造成細胞生存能力的降低。Caspase 3/7 活力隨著膀胱癌細胞以 MMC 或靜水壓的處理而增加。以10 kPa 靜水壓合併處理 MMC 可引起 FasL 的表現力的增加。於 10 μg/mL MMC 以及 10 kPa 靜水壓的合併處理下,膀胱癌細胞粒線體表現出增加膜電位差的受損。
上述結果顯示靜水壓可協同加強 MMC 引起膀胱癌細胞凋亡是經由 Fas/FasL 途徑的現象。為了進一步瞭解膀胱癌細胞因為靜水壓以及 MMC 引起基因表現的改變,本文使用微陣列 (oligonucleotide microarray) 分析所有基因表現的差異。在經由生物資訊學的分析和基因候選,以及經由即時聚合酶鏈鎖反應 (real-time PCR) 以及免疫墨點法 (Immunoblotting) 確定後,類Toll受體 (TLR6) 和結締組織生長因子 (CTGF) 於基因的改變表現出顯著之上調控差異。因此本文總結在以靜水壓以及 MMC 的合併處理下,膀胱癌細胞顯示出增加細胞凋零的現象是經由 TLR6 和 CTGF 上調控引起外部途徑所造成。
摘要(英) Abstract
Urothelial carcinoma (UC) of the bladder is the second most common cancer of the genitourinary system. UC is the most common histologic subtype of bladder cancer. Clinical UC treatment usually involves transurethral resection of the bladder tumor followed by adjuvant intravesical immunotherapy or chemotherapy to prevent recurrence. Intravesical chemotherapy induces fewer side effects than immunotherapy but is less effective at preventing tumor recurrence. The administration of mitomycin C into bladder after TURBT is a common treatment strategy for preventing recurrence after surgery. Improvement to intravesical chemotherapy is, therefore, clinically needed.
This study investigates the possibility of applying hydrostatic pressure to augment the effectiveness of MMC treatment on preventing UC cells recurrence. The cell lineage BFTC905 cells were assessed. Viability of the UC cells was determined using cellular proliferation assay. Flow cytometry was used to determined the apoptosis. Changes in apoptotic function were evaluated by caspase 3/7 activities, expression of FasL, and loss of mitochondrial membrane potential. Reduced cell viability was associated with increasing hydrostatic pressure. Caspase 3/7 activities were increased following treatment of the UC cells with MMC or hydrostatic pressure. In combination with 10 kPa hydrostatic pressure, MMC treatment induced increasing FasL expression. The mitochondria of UC cells displayed increasingly impaired membrane potentials following combined treatment with 10 μg/mL MMC and 10 kPa hydrostatic pressure.
Hydrostatic pressure combined with MMC in UC cells was found to synergistically enhanced MMC-induced UC cell apoptosis via the Fas/FasL pathways. To understand the alteration of gene expressions in UC cells caused by hydrostatic pressure and MMC, oligonucleotide microarray was used to explore all of the differentially expressed genes. After bioinformatics analysis and gene annotation, Toll-like receptor 6 and connective tissue growth factor showed significant up-regulation among altered genes, and their expressions with each treatment of UC cells were validated by real-time PCR and Immunoblotting. We conclude that under treatment with MMC and hydrostatic pressure, UC cells showed increasing apoptosis via extrinsic pathways through upregulation of TLR6 and CTGF.
關鍵字(中) ★ 泌尿上皮細胞癌
★ 絲裂霉素C
★ 經尿道內視鏡腫瘤切除術
★ 即時聚合酶鏈鎖反應
★ 類Toll受體
★ 結締組織生長因子
關鍵字(英) ★ UC, urothelial carcinoma
★ MMC, mitomycin C
★ TURBT, transurethral resection of the bladder tumor
★ BCG, Bacillus Calmette- Guerin
★ TC, Thermo-chemotherapy
★ EMDA, Electromotive drug administration
論文目次 第一章 緒論………………………………………………………… 16
1.1. 膀胱癌的簡介與治療………………………………………… 18
1.2. 文獻回顧……………………………………………………… 39
1.2.1 增加絲裂霉素C 膀胱灌注治療功效的方法………………39
1.2.2機械應力運用於細胞生長及分化的研究………………………44
1.2.3 絲裂霉素C 治療子宮頸癌效果的研究………………………46
1.2.4 細胞凋亡的簡介…………………………………………………46
第二章 研究動機及假設(I)………………………………………… 52
第三章 實驗設計(I)…………………………………………………54
3.1. 材料與方法……………………………………………………54
3.1.1 靜水壓生物反應器……………………………………………54
3.1.2 膀胱癌細胞株施壓方示………………………………………55
3.1.3細胞生存能力的測定…………………………………………56
3.1.4 統計分析…………………………………………………………57
3.2. 初步結果與討論…………………………………………………57
第四章 細胞凋亡的實驗………………………………………………63
4.1. 材料與方法……………………………………………………… 63
4.1.1凋亡細胞亞二倍體分析……………………………………63
4.1.2 細胞凋亡蛋白酶 Caspase 3/7 活動力的分析 ……………65
4.1.3 即時聚合酶鏈鎖反應…………………………………………66
4.1.4 西方墨點法…………………………………………………67
4.1.5 細胞凋亡活動力及粒線體膜電位變化…………………67
4.2. 結果……………………………………………………………68
4.2.1凋亡細胞亞二倍體分析………………………………………68
4.2.2 靜水壓和絲裂霉素C濃度對 caspase 3/7 活動力的影響 ……69
4.2.3 靜水壓和 MMC 處理造成細胞凋亡的協同作用……………69
4.2.4 細胞凋亡活動力及粒線體膜電位變化………………………70
4.3. 討論………………………………………………………………70
第五章 研究動機假設及實驗設計(II)………………………………78
第六章 基因表達的輪廓…………………………………………79
6.1. 材料與方法………………………………………………………79
6.1.1 膀胱癌細胞總核醣核酸的萃取…………………………………79
6.1.2 微陣列融合 ………………………………………………80
6.1.3 陣列資料分析………………………………………………80
6.1.4 反轉轉錄及基因表現的確認…………………………………81 6.1.5 TLR6 and CTGF蛋白質免疫墨點法………………………82
6.1.6 統計分析 ………………………………………………………83
6.2. 結果………………………………………………………………84
6.2.1 絲裂霉素C處理膀胱癌細胞對靜水壓有反應候選者的選擇…84
6.2.2 不同基因表現路徑以及蛋白質互相作用……………………84
6.2.3即時聚合酶鏈鎖反應及免疫墨點法結果………………………85
6.3. 討論………………………………………………………………86
第七章 結論與未來展望………………………………………………94
參考文獻……………………………………………………………… 96
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余明翰,2012,靜水壓對細胞分化及增生影響的實驗研究,中央大學機械工程學系碩士論文。
指導教授 鍾志昂(Chih-Ang Chung) 審核日期 2014-7-28
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