氣壓影響膀胱癌細胞之分子機制探討

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呂登貴(Teng-Guei Leu) 查詢紙本館藏

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機械工程學系

論文名稱

氣壓影響膀胱癌細胞之分子機制探討

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摘要(中)

癌細胞發展為惡性腫瘤的原因，在於其細胞中的細胞週期素 (cyclins) 過度表達或是細胞週期素依賴性激酶抑制物 (CKIs) 的缺失或是腫瘤抑制因子的突變與缺失，進而導致細胞週期內的檢查點監控機制失效，使得癌細胞獲得抵抗凋亡的能力，癌細胞也因此得到抗凋亡的能力。膀胱癌為人類泌尿系統中常見的腫瘤之一，對於膀胱癌之治療方案，一般採用手術後輔以膀胱灌注法之方式，將藥物直接灌注至病患膀胱中，透過藥物徹底殺死癌細胞，以避免腫瘤復發。灌注藥物期間患者膀胱內的壓力也會提高，此種壓力刺激可能具有抑制細胞增生的效果，並且也可配合藥物刺激以提升誘導膀胱癌細胞凋亡的效率，但仍不清楚此種機械應力對膀胱癌細胞的生理機制有何影響。
本研究探討氣壓對於膀胱癌細胞之分子機制，以氣壓式生物反應器對BFTC905膀胱癌細胞株施以壓力刺激，並透過MTT assay證實，以40 kPa之壓力刺激10小時之後處於G1期之細胞量有比例有明顯的增加，說明靜水壓刺激會誘導細胞週期於G1期停滯。同時也發現位於G1初期的cyclin D、CDK4與CDK6的表現量上升，但是位於G1末期的cyclin E與CDK2的表現量卻下降，這也可以說明細胞週期在G1初期即受到抑制。另外由流式細胞儀分析中可以得知，處於S期、G2期與M期之細胞量比例隨著加壓的時間上升而逐漸變少，也許是因為大部分的細胞都停被抑制在G1期的緣故，因而導致其他時期的細胞量比例相對減少。
此外p27kip1在經過壓力刺激10小時之後表現量有大幅地上升，而p21Cip1/waf1則僅有些微的增加，接著我們也發現Rb於Ser608的低磷酸化，這些證據說明了壓力刺激將提高p21Cip1/waf1與p27kip1的表現，抑制cyclin E1-CDK2並且間接影響了Rb於Ser608的磷酸化程度以誘導G1停滯。

摘要(英)

Tumor cells develop owing to overexpression of cyclins or lack of CKIs. These mutations result in the fault of regulatory checkpoints mechanism of cell cycle, whereby cancer cells obtain the abilities to resist apoptosis. Bladder cancer is a common cancer in the human urinary system. As a companion to the surgical treatment, patients can be also treated by the assistant intravesical therapy that utilizes chemotherapy agents such as mytomicin C to kill the remaining cancer cells after the surgery to reduce tumor recurrent. During the treatment of chemotherapy, the pressure influence should be considered as a factor that affects the intravesical condition. Previous research has found that mytomicin C induced apoptosis is enhanced by applying the static pressure, suggesting that pressure treatment has the ability to inhibit cancer cell proliferation. However, the cellular mechanism of this mechanical stress still remains unclear.
In this study we develop and utilize a gas pressure bioreactor system to impose static pressure (40 kPa) on BFTC905 cells, a bladder cancer cell line, and investigate how this mechanical stress affects the bladder cancer cells. Results from MTT assay showed that the cell proliferation was inhibited by the static pressure treatment. Results from flow cytometry indicated that a large portion of cells were arrested in G1 phase after pressure application for 10hours. Western blots analysis showed that the protein expressions of cyclin D (D1, D3), CDK4 and CDK6, which regulate early G1 phase cell cycle, were up-regulated compared with the controlled groups after pressure treatment in BFTC905 cells. We also found that the expressions of cyclin E1 and CDK2, which regulate late G1 phase cell cycle, were down-regulated after pressure treatment. The percentage of cell population gradually decreased in S-phase, G2-phase and M-phase relative to G1-phase over the pressure treatment, probably due to G1-phase arrest caused by static pressure.
Furthermore we found that the cyclin-dependent-kinase inhibitor p27kip1 was up-regulated profoundly after 10hr pressure treatment, and p21Cip1/waf1 was slightly increased. This implied that the role played by p27kip1 may be more important than p21Cip1/waf1 in static pressure treatment. Also, dephosphorylation of Ser608 in Rb after the pressure treatment confirmed that the cell cycle was arrested in G1-phase. According to these findings, we concluded that the static pressure induced cell cycle arrest at G1-phase through escalating the p21 Cip1/waf1 and p27 Cip1/waf1 levels, decreasing the cyclin E1 and CDK2 expressions, and inducing the dephosphorylation of Ser608 in Rb.

關鍵字(中)

★ 膀胱癌細胞
★ 壓力
★ 細胞週期
★ G1停滯

關鍵字(英)

★ bladder cancer
★ pressure
★ cell cycle
★ G1 arrest

論文目次

摘要 i
致謝 iv
目錄 v
第一章緒論
1.1. 研究動機 1
1.2. 文獻回顧 2
1.2.1. 細胞週期 2
1.2.2. 細胞週期素 3
1.2.3. 細胞週期素調控蛋白- p21Cip1/waf 與p27kip1 6
1.2.4. 壓力式生物反應器 7
1.3. 研究目的 8
第二章實驗設計與方法 9
2.1. 氣壓式生物反應器 9
2.1.1. 實驗環境設置與步驟 10
2.1.2. 生物反應器構造 11
2.2. 細胞培養 12
2.2.1. 解凍細胞 13
2.2.2. 細胞繼代 (Passage) 13
2.2.3. 細胞冷凍保存 13
2.2.4. 細胞計數 14
2.3. MTT assay與MTT比色法 14
2.4. 實驗裝置與壓力刺激實驗 15
2.5. 流式細胞儀分析 (Flow cytometric analysis) 16
2.5.1. 樣本製備 16
2.5.2. 實驗操作 17
2.6. 蛋白質定量分析 17
2.6.1. 樣品處理 17
2.6.2. 定量分析 17
2.7. 蛋白質體電泳 (SDS-PAGE) 18
2.7.1. 架設凝膠裝置 18
2.7.2. 配製聚丙醯胺之分離膠體溶液(下層焦集膠體溶液)18
2.7.3. 配製聚丙醯胺之分離膠體溶液(上層焦集膠體溶液)19
2.7.4. 配製電泳緩衝溶液 (running buffer) 19
2.7.5. 蛋白質樣品處理 19
2.7.6. 電泳操作 20
2.8. 西方墨點分析法 (Western Blot analysis) 20
2.8.1. SDS-PAGE 20
2.8.2. 半濕式 (semi-dry) 蛋白質轉印法 20
2.8.3. 免疫染色 21
2.8.4. ECL (enhance chemiluminescent reagent) 冷光呈色 21
第三章結果與討論 23
3.1. 生物相容性測試 23
3.2. 氣壓對細胞生長速率的影響 25
3.3. 壓力對細胞週期的影響 26
3.4. 蛋白質表現量 29
3.4.1. G1前期之細胞週期相關的蛋白質表現量變化 30
3.4.2. G1末期之細胞週期相關的蛋白質表現量變化 31
3.4.3. S期之細胞週期相關的蛋白質表現量變化 34
3.4.4. G2期之細胞週期相關的蛋白質表現量變化 35
3.4.5. M期之細胞週期相關的蛋白質表現量變化 37
3.4.6. p21Cip1/waf1與p27kip1之蛋白質表現量變化 38
第四章結論與未來望 40
參考文獻 42

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指導教授

鍾志昂

審核日期

2016-8-25

推文