設計與製作圓錐平板型生物反應器並用以探討流體剪應力對膀胱癌細胞自噬行為之影響

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林楷恒(Kai-Heng Lin) 查詢紙本館藏

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論文名稱

設計與製作圓錐平板型生物反應器並用以探討流體剪應力對膀胱癌細胞自噬行為之影響

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摘要(中)

已有許多研究指出機械力刺激細胞在細胞的型態、功能以及訊息傳遞上扮演重要的角色，而這些機械力同樣也與許多疾病的發展有著重要的關聯。有鑑於物理性刺激研究需要有專門的生物反應器來提供細胞機械力刺激，因此本論文第一部份是以人因工程為導向設計出圓錐平板型生物反應器並用以探討流體剪應力對膀胱癌細胞的影響。此全新圓錐平板型生物反應器特點除了可以提供穩定、均勻的流體剪應力外，也具備維修容易、操作技術門檻低、適用於市售培養皿等優點。從細胞相容性測試、細胞活性分析以及西方墨點法的實驗結果來看，皆證實了本文所設計的生物反應器不但可以作後續的生物實驗分析，也可以重現他人的研究成果，這代表新設計的裝置可以提供穩定的實驗條件。本論文的第二部份為利用此新的生物反應器進行流體剪應力刺激膀胱癌細胞後細胞內之生理反應的研究。由我們的實驗結果發現對膀胱癌細胞株BFTC-905施予12 dynes/cm2 的流體剪應力並刺激24小時，在結合細胞自噬抑制劑的實驗條件下，能拯救細胞的活性；另外，細胞染色螢光訊號的增強也說明了受到流體剪應力刺激過後，細胞會產生自噬行為來回應。透過西方墨點法分析我們觀察到了LC3BII的上調控，Beclin-1、caspase-3、8、9以及PARP於其截切位置均未發現被截切的片段。這些證據證實了承受所施加強度之流體剪應力刺激過後的膀胱細胞會藉由自噬行為嘗試渡過所遇到的逆境，而這段過程並不會誘導細胞凋亡也不會從細胞自噬轉向細胞凋亡，因此本論文認為流體剪應力對於細胞的生理意義為細胞自噬的誘導因子。

摘要(英)

Mechanical force stimulation plays an important role not only in biological morphology, functions and signal transductions but also associated with development of numerous diseases. In order to apply the physical stimulation, the first part of this thesis is based on human factors engineering as a guide to design a cone-plate bioreactor to investigate the shear-stress influence on the stimulated bladder cancer cells. This novel cone-plate bioreactor can provide stable and uniform fluid shear stress and possess advantages such as easy maintenance, low operation barrier and being-ready for commercially available petri dishes. Experiment results of cell compatibility test, cell viability analysis and Western blot verified that this bioreactor not only can be used for subsequent biological experimental analyses. By repeating the research results available in literature we showed this bioreactor can supply stable experimental conditions continuously. The second part of this paper is to study the physiological responses of bladder cancer cells stimulated by fluid shear stresses using this new bioreactor. Experimental results show that when the bladder cancer cell line BFTC-905 was subjected to 12 dynes/cm2 fluid shear stress for 24 hours, the cells’ viability declined. The application of autophagy inhibitor could rescue the cell viability. The enhancement of cell-stained fluorescent signal also illustrated autophagic response occurrence after cells were stimulated by fluid shear stress. Through the Western blot analysis we observed the LC3BII was up-regulated, the cleaved fragments of Beclin-1, caspase-3,8,9 and PARP were not found at its cleaved status. These evidences proved that the bladder cancer cell tried to overcome its adversity by taking the autophagic pathway when subjected to fluid shear stress. The shear stress neither induced apoptosis nor transition from autophagy to apoptosis. We concluded the fluid shear stress was an autophagic inducer for the physiological meaning of cell.

關鍵字(中)

★ 圓錐平板型生物反應器
★ 流體剪應力
★ 膀胱癌細胞
★ 細胞自噬

關鍵字(英)

論文目次

摘要 i
Abstract ii
謝誌 iii
目錄 v
圖目錄 viii
表目錄 xi
第一章緒論 1
1.1 研究動機 1
1.2 文獻回顧 3
1.2.1 膀胱癌與治療方法 3
1.2.2 細胞凋亡與自噬 4
1.2.3 流體剪應力刺激對細胞的影響 5
1.2.4 流體剪應力型生物反應器 7
1.3 研究目的 9
第二章生物反應器設計與製作 13
2.1 設計前言 13
2.2 流體剪應力 16
2.2.1 流體剪應力產生原理 16
2.2.2 流體剪應力計算 16
2.2.3 圓錐設計 18
2.2.4 培養液性質量測 18
2.2.5 系統性能表現 19
2.3 圓錐平板生物反應器機構設計與組裝 19
2.3.1 培養皿治具設計 19
2.3.2 扭力量測機構設計 20
2.3.3 底盤機構設計 22
2.3.4 傳動機構設計 22
2.3.5 圓錐驅動裝置設計 23
2.3.6 扭力量測機構驗證 25
2.4 細胞培養環境系統設計與製作 25
2.4.1 細胞培養條件 26
2.4.2 箱體設計與系統控制迴路 26
2.4.3 均溫性測試 29
第三章實驗方法 49
3.1 細胞培養相關操作 50
3.2 流體剪應力刺激實驗 51
3.3 細胞活性分析 (MTT assay) 52
3.4 螢光染色分析 53
3.5 西方墨點法 (Western Blot analysis) 54
3.5.1 蛋白質定量分析 54
3.5.2 蛋白質電泳 (SDS-PAGE) 55
3.5.3 半濕式 (semi-dry) 蛋白質轉印法 57
3.5.4 免疫染色 57
3.6 統計定量 59
第四章結果與討論 60
4.1 細胞活性分析實驗結果 60
4.1.1 單純流體剪應力刺激 61
4.1.2 流體剪應力刺激結合自噬抑制劑 61
4.1.3 流體剪應力刺激結合凋亡抑制劑 62
4.2 細胞自噬相關之螢光染色分析實驗結果 62
4.3 西方墨點法實驗結果 63
4.3.1 流體剪應力刺激對細胞自噬相關蛋白質的表現量變化 63
4.3.2 流體剪應力刺激對細胞凋亡相關蛋白質的表現量變化 64
4.3.3 流體剪應力刺激對其他蛋白質的表現量變化 64
4.4 討論 65
第五章結論與未來展望 79
參考文獻 82
附錄 90

參考文獻

Akimoto, S., Mitsumata, M., Sasaguri, T., & Yoshida, Y. (2000). Laminar shear stress inhibits vascular endothelial cell proliferation by inducing cyclin-dependent kinase inhibitor p21Sdi1/Cip1/Waf1. Circulation Research, 86(2), 185-190.
Alberts B., Johnson A., Lewis J.., Raff M., Roberts K.. & Walter., Molecular biology of the cell, Garland Science., USA, 2002
Amé, J. C., Spenlehauer, C., & de Murcia, G. (2004). The PARP superfamily. Bioessays, 26(8), 882-893.
Bacac, M., & Stamenkovic, I. (2008). Metastatic cancer cell. Annu. Rev. pathmechdis. Mech. Dis., 3, 221-247.
Baker, S. J., Fearon, E. R., Nigro, J. M., Hamilton, S. R., Preisinger, A. C., Jessup, J. M., & White, R. (1989). Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science, 244(4901), 217-221.
Blackman, B. R., Garcıa-Cardena, G., & Gimbrone, M. A. (2002). A new in vitro model to evaluate differential responses of endothelial cells to simulated arterial shear stress waveforms. Journal of biomechanical engineering, 124(4), 397-407. Borden, Lester S., Peter E. Clark, and M. Craig Hall. ”Bladder cancer.” Current opinion in oncology 15.3 (2003): 227-233.
Bouchard, V. J., Rouleau, M., & Poirier, G. G. (2003). PARP-1, a determinant of cell survival in response to DNA damage. Experimental hematology, 31(6), 446-454.
Boulares, A. H., Yakovlev, A. G., Ivanova, V., Stoica, B. A., Wang, G., Iyer, S., & Smulson, M. (1999). Role of poly (ADP-ribose) polymerase (PARP) cleavage in apoptosis Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells. Journal of Biological Chemistry, 274(33), 22932-22940.

Brausi, M., Oddens, J., Sylvester, R., Bono, A., van de Beek, C., van Andel, G., & Oosterlinck, W. (2014). Side effects of Bacillus Calmette-Guerin (BCG) in the treatment of intermediate-and high-risk Ta, T1 papillary carcinoma of the bladder: results of the EORTC genito-urinary cancers group randomised phase 3 study comparing one-third dose with full dose and 1 year with 3 years of maintenance BCG. European urology, 65(1), 69-76.
Breen, L. T., McHugh, P. E., McCormack, B. A., Muir, G., Quinlan, N. J., Heraty, K. B., & Murphy, B. P. (2006). Development of a novel bioreactor to apply shear stress and tensile strain simultaneously to cell monolayers. Review of scientific instruments, 77(10), 104301.
Brown, Matthew F. ”Caspase-3 promotes cell proliferation and inhibits dna-damage induced necrosis in colorectal cancer.” Diss. University of Pittsburgh, 2013.
Cengel Y. A., Cimbala J. M., fluid mechanics: fundamentals and applications, McGraw-Hill, New York, 2006.
Chang, S. F., Chang, C. A., Lee, D. Y., Lee, P. L., Yeh, Y. M., Yeh, C. R., & Chiu, J. J. (2008). Tumor cell cycle arrest induced by shear stress: Roles of integrins and Smad. Proceedings of the National Academy of Sciences, 105(10), 3927-3932.
Cheng, D. H. (1968). The effect of secondary flow on the viscosity measurement using the cone-and-plate viscometer. Chemical Engineering Science, 23(8), 895-899.
Cheng, Y. T., Li, Y. L., Wu, J. D., Long, S. B., Tzai, T. S., Tzeng, C. C., & Lai, M. D. (1995). Overexpression of MDM‐2 mRNA and mutation of the p53 tumor suppressor gene in bladder carcinoma cell lines. Molecular carcinogenesis, 13(3), 173-181.
Chien, S. (2007). Mechanotransduction and endothelial cell homeostasis: the wisdom of the cell. American Journal of Physiology-Heart and Circulatory Physiology, 292(3), H1209-H1224.
Cohen, G. M. (1997). Caspases: the executioners of apoptosis. Biochemical Journal, 326(1), 1-16.

Colombel, M., Soloway, M., Akaza, H., Böhle, A., Palou, J., Buckley, R., & Persad, R. (2008). Epidemiology, staging, grading, and risk stratification of bladder cancer. european urology supplements, 7(10), 618-626.
Cookson, M. S., Herr, H. W., Zhang, Z. F., Soloway, S., Sogani, P. C., & Fair, W. R. (1997). The treated natural history of high risk superficial bladder cancer: 15-year outcome. The Journal of urology, 158(1), 62-67.
Cox, D. B. (1962). Radial flow in the cone-plate viscometer. Nature, 193(4816), 670-670.
Davis, C. A., Zambrano, S., Anumolu, P., Allen, A. C., Sonoqui, L., & Moreno, M. R. (2015). Device-based in vitro techniques for mechanical stimulation of vascular cells: a review. Journal of biomechanical engineering, 137(4), 040801.
Denicourt, C., & Dowdy, S. F. (2004). Cip/Kip proteins: more than just CDKs inhibitors. Genes & development, 18(8), 851-855.
DePaola, N., Gimbrone, M. A., Davies, P. F., & Dewey, C. F. (1992). Vascular endothelium responds to fluid shear stress gradients. Arteriosclerosis, Thrombosis, and Vascular Biology, 12(11), 1254-1257.
Dewey, C. F., Bussolari, S. R., Gimbrone, M. A., & Davies, P. F. (1981). The dynamic response of vascular endothelial cells to fluid shear stress. Journal of biomechanical engineering, 103(3), 177-185.
Eisenberg-Lerner, A., Bialik, S., Simon, H. U., & Kimchi, A. (2009). Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell death and differentiation, 16(7), 966.
Elmore, S. (2007). Apoptosis: a review of programmed cell death. Toxicologic pathology, 35(4), 495-516.
Einav, S., Dewey, C. F., & Hartenbaum, H. (1994). Cone-and-plate apparatus: a compact system for studying well-characterized turbulent flow fields. Experiments in fluids, 16(3), 196-202.
Fesik, S. W. (2005). Promoting apoptosis as a strategy for cancer drug discovery. Nature reviews. Cancer, 5(11), 876.
Green, D. R., & Kroemer, G. (2009). Cytoplasmic functions of the tumor suppressor p53. Nature, 458(7242), 1127.
Hanahan, D., & Weinberg, R. A. (2011). Hallmarks of cancer: the next generation. cell, 144(5), 646-674.
Hoffman, B. D., Grashoff, C., & Schwartz, M. A. (2011). Dynamic molecular processes mediate cellular mechanotransduction. Nature, 475(7356), 316.
Hollstein, M., Sidransky, D., Vogelstein, B., & Harris, C. C. (1991). p53 mutations in human cancers. Science, 253(5015), 49-53.
Javle, M., & Curtin, N. J. (2011). The role of PARP in DNA repair and its therapeutic exploitation. British journal of cancer, 105(8), 1114.
Kang, R., Zeh, H. J., Lotze, M. T., & Tang, D. (2011). The Beclin 1 network regulates autophagy and apoptosis. Cell death and differentiation, 18(4), 571.
Khan, M., Maryam, A., Zhang, H., Mehmood, T., & Ma, T. (2016). Killing cancer with platycodin D through multiple mechanisms. Journal of cellular and molecular medicine, 20(3), 389-402.
Kimura, S., Fujita, N., Noda, T., & Yoshimori, T. (2009). Monitoring autophagy in mammalian cultured cells through the dynamics of LC3. Methods in enzymology, 452, 1-12.
Lamkanfi, M., Festjens, N., Declercq, W., Berghe, T. V., & Vandenabeele, P. (2007). Caspases in cell survival, proliferation and differentiation. Cell death and differentiation, 14(1), 44.
Lee, Y. J., Ha, Y. J., Kang, Y. N., Kang, K. J., Hwang, J. S., Chung, W. J., & Kim, M. K. (2013). The autophagy-related marker LC3 can predict prognosis in human hepatocellular carcinoma. PloS one, 8(11), e81540.

Lien, S. C., Chang, S. F., Lee, P. L., Wei, S. Y., Chang, M. D. T., Chang, J. Y., & Chiu, J. J. (2013). Mechanical regulation of cancer cell apoptosis and autophagy: roles of bone morphogenetic protein receptor, Smad1/5, and p38 MAPK. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1833(12), 3124-3133.
Lockshin, R. A., & Zakeri, Z. (2004). Apoptosis, autophagy, and more. The international journal of biochemistry & cell biology, 36(12), 2405-2419.
Luo, C. W., Wu, C. C., & Ch′ang, H. J. (2014). Radiation sensitization of tumor cells induced by shear stress: The roles of integrins and FAK. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1843(9), 2129-2137.
Martin, I., Wendt, D., & Heberer, M. (2004). The role of bioreactors in tissue engineering. TRENDS in Biotechnology, 22(2), 80-86.
Martini F. H., Nath J. L., & Bartholomew E. F., Fundamentals of anatomy & physiology, Pearson Benjamin Cummings. San Francisco, 2006.
Mitra, A. P., Datar, R. H., & Cote, R. J. (2006). Molecular pathways in invasive bladder cancer: new insights into mechanisms, progression, and target identification. Journal of Clinical Oncology, 24(35), 5552-5564.
Mizushima, N. (2004). Methods for monitoring autophagy. The international journal of biochemistry & cell biology, 36(12), 2491-2502.
Mizushima, N., Yoshimori, T., & Levine, B. (2010). Methods in mammalian autophagy research. Cell, 140(3), 313-326.
Mooney, M., & Ewart, R. H. (1934). The conicylindrical viscometer. Physics, 5(11), 350-354.
Nakahira, K., & Choi, A. M. (2013). Autophagy: a potential therapeutic target in lung diseases. American Journal of Physiology-Lung Cellular and Molecular Physiology, 305(2), L93-L107.
Nerem, R. M. (2006). Tissue engineering: the hope, the hype, and the future. Tissue engineering, 12(5), 1143-1150.
Nigro, J. M., Baker, S. J., Preisinger, A. C., Jessup, J. M., Hosteller, R., Cleary, K., & Glover, T. (1989). Mutations in the p53 gene occur in diverse human tumour types. Nature, 342(6250), 705-708.
Ott, O. J., Rödel, C., Weiss, C., Wittlinger, M., Krause, F. S., Dunst, J., & Sauer, R. (2009). Radiochemotherapy for bladder cancer. Clinical Oncology, 21(7), 557-565.
Ozturk S., Hu W. S., Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, CRC Press., UK, 2005.
Paglin, S., Hollister, T., Delohery, T., Hackett, N., McMahill, M., Sphicas, E., & Yahalom, J. (2001). A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer research, 61(2), 439-444.
Panda, P. K., Mukhopadhyay, S., Das, D. N., Sinha, N., Naik, P. P., & Bhutia, S. K. (2015). Mechanism of autophagic regulation in carcinogenesis and cancer therapeutics. In Seminars in cell & developmental biology (Vol. 39, pp. 43-55). Academic Press.
Pelech, I., & Shapiro, A. H. (1964). Flexible disk rotating on a gas film next to a wall. Journal of Applied Mechanics, 31(4), 577-584.
Riley, T., Sontag, E., Chen, P., & Levine, A. (2008). Transcriptional control of human p53-regulated genes. Nature reviews. Molecular cell biology, 9(5), 402.
Sdougos, H. P., Bussolari, S. R., & Dewey, C. F. (1984). Secondary flow and turbulence in a cone-and-plate device. Journal of Fluid Mechanics, 138, 379-404.
Spiess, P. E., & Czerniak, B. (2006). Dual-track pathway of bladder carcinogenesis: practical implications. Archives of pathology & laboratory medicine, 130(6), 844-852.
Spruell, C., & Baker, A. B. (2013). Analysis of a high‐throughput cone‐and‐plate apparatus for the application of defined spatiotemporal flow to cultured cells. Biotechnology and bioengineering, 110(6), 1782-1793.
Stegemann, J. P., Hong, H., & Nerem, R. M. (2005). Mechanical, biochemical, and extracellular matrix effects on vascular smooth muscle cell phenotype. Journal of applied physiology, 98(6), 2321-2327.
Stolberg, S., & McCloskey, K. E. (2009). Can shear stress direct stem cell fate?. Biotechnology progress, 25(1), 10-19.
Sucosky, P., Padala, M., Elhammali, A., Balachandran, K., Jo, H., & Yoganathan, A. P. (2008). Design of an ex vivo culture system to investigate the effects of shear stress on cardiovascular tissue. Journal of biomechanical engineering, 130(3), 035001.
Sylvester, R. J. (2006). Natural history, recurrence, and progression in superficial bladder cancer. The Scientific World Journal, 6, 2617-2625.
Sun, Y., & Peng, Z. L. (2009). Programmed cell death and cancer. Postgraduate medical journal, 85(1001), 134-140.
Takahashi, T., Nau, M. M., Chiba, I., Birrer, M. J., & Rosenberg, R. K. (1989). p53: a frequent target for genetic abnormalities in lung cancer. Science, 246(4929), 491.
Vogan, K., Bernstein, M., Leclerc, J. M., Brisson, L., Brossard, J., Brodeur, G. M., & Gros, P. (1993). Absence of p53 gene mutations in primary neuroblastomas. Cancer research, 53(21), 5269-5273.
Walsh, J. G., & Martin, S. J. (2010). Caspases, Substrates and Sequential Activation. eLS.
Walters, K., Waters, N.D., 1966. Polymer systems, deformation and flow. In Proc. Brit. Soc. Rheol. (ed. R. E. Wetton and R. W. Whorlow). Macmmillan.
Wang, K. C., Garmire, L. X., Young, A., Nguyen, P., Trinh, A., Subramaniam, S., & Chien, S. (2010). Role of microRNA-23b in flow-regulation of Rb phosphorylation and endothelial cell growth. Proceedings of the National Academy of Sciences, 107(7), 3234-3239.
Watson J. D., Molecular Biology of the Gene, Pearson Benjamin Cummings. San Francisco, 2008.
Wirawan, E., Walle, L. V., Kersse, K., Cornelis, S., Claerhout, S., Vanoverberghe, I., & Agostinis, P. (2010). Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria. Cell death & disease, 1(1), e18.
Yee, K. S., & Vousden, K. H. (2005). Complicating the complexity of p53. Carcinogenesis, 26(8), 1317-1322.
NCI, National Cancer Institue, What is cancer?. 2015.
取自https://www.cancer.gov/about-cancer/understanding/what-is-cancer#types-of-cancer
WHO, World Health Organization, Cancer., 2017.
取自http://www.who.int/mediacentre/factsheets/fs297/en/
王文甫，設計與製作圓錐平板型生物反應器，碩士論文，國立中央大學機械工程學系，2008
馬大翔，設計與製作圓錐平板型生物反應器以探討剪應力對大鼠骨髓幹細胞生長與型態之影響，碩士論文，國立中央大學機械工程學系，2009
劉炫志，設計與製作圓錐平板型生物反應器以探討剪應力對幹細胞生長與型態之影響，碩士論文，國立中央大學機械工程學系，2009
劉威宏，流體剪應力結合1-甲基-3-異丁基黃嘌呤對於人類胎盤幹細胞分化之影響，碩士論文，國立中央大學機械工程學系，2012
施秉玠，靜水壓影響膀胱癌細胞之分子機制探討，碩士論文，國立中央大學機械工程學系，2013
陳紹寬，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，博士論文，國立中央大學機械工程學系，2014
郭弘偉，直接電刺激對於人類牙髓幹細胞在骨分化過程中基因調控與分化能力影響之研究，碩士論文，國立中央大學化學工程與材料工程學系，2015
游天賜，流體剪應力對膀胱癌細胞周期的影響，碩士論文，國立中央大學機械工程學系，2016
臺灣衛生福利部國民健康署，癌症登記報告，2016

指導教授

鍾志昂

審核日期

2017-8-15

推文