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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 侯詠德(Yung-Te Hou) | |
| dc.contributor.author | Ying-Hsiu Liao | en |
| dc.contributor.author | 廖英秀 | zh_TW |
| dc.date.accessioned | 2021-06-17T01:15:18Z | - |
| dc.date.available | 2022-08-19 | |
| dc.date.copyright | 2020-08-21 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-19 | |
| dc.identifier.citation | 1.李俊諺。2018。標靶藥物載體之劑型開發用於肝組織再生之研究。碩士論文。台北: 台灣大學生物產業機電工程研究所。 2.Almazroo, O. A., Miah, M. K., Venkataramanan, R. (2017). Drug Metabolism in the Liver. Clinics in Liver Disease, 21(1), 1–20. 3.American Pharmaceutical Association. (1996). Handbook of nonprescription drugs. American Pharmaceutical Association. 4.Au, S. H., Chamberlain, M. D., Mahesh, S., Sefton, M. V., Wheeler, A. R. (2014). Hepatic organoids for microfluidic drug screening. Lab on a Chip, 14(17), 3290. 5.Avril, A., Pichard, V., Bralet, M.-P., Ferry, N. (2004). Mature hepatocytes are the source of small hepatocyte-like progenitor cells in the retrorsine model of liver injury. Journal of Hepatology, 41(5), 737–743. 6.Bale, S. S., Moore, L., Yarmush, M., Jindal, R. (2016). EmergingIn VitroLiver Technologies for Drug Metabolism and Inter-Organ Interactions. Tissue Engineering Part B: Reviews, 22(5), 383–394. 7.Beckwitt, C. H., Clark, A. M., Wheeler, S., Taylor, D. L., Stolz, D. B., Griffith, L., Wells, A. (2018). Liver ‘organ on a chip.’ Experimental Cell Research, 363(15–25). 8.Bhise, N. S., Manoharan, V., Massa, S., Tamayol, A., Ghaderi, M., Miscuglio, M., Lang, Q., Shrike Zhang, Y., Shin, S. R., Calzone, G., Annabi, N., Shupe, T. D., Bishop, C. E., Atala, A., Dokmeci, M. R., Khademhosseini, A. (2016). A liver-on-a-chip platform with bioprinted hepatic spheroids. Biofabrication, 8(1), 014101. 9.Carrel, A. (1912). PURE CULTURES OF CELLS. The Journal of Experimental Medicine, 16(2), 165–168. 10.Chen, A. A., Khetani, S. R., Lee, S., Bhatia, S. N., Van Vliet, K. J. (2009). Modulation of hepatocyte phenotype in vitro via chemomechanical tuning of polyelectrolyte multilayers. Biomaterials, 30(6), 1113–1120. 11.Chen, Q., Kon, J., Ooe, H., Sasaki, K., Mitaka, T. (2007). Selective proliferation of rat hepatocyte progenitor cells in serum-free culture. Nature Protocols, 2(5), 1197–1205. 12.Chen, Y.-H., Chang, M.-H., Chien, C.-S., Wu, S.-H., Yu, C.-H., Chen, H.-L. (2013). Contribution of mature hepatocytes to small hepatocyte-like progenitor cells in retrorsine-exposed rats with chimeric livers. Hepatology, 57(3), 1215–1224. 13.Cheng, J.-Y., Wei, C.-W., Hsu, K.-H., Young, T.-H. (2004). Direct-write laser micromachining and universal surface modification of PMMA for device development. Sensors and Actuators B: Chemical, 99(1), 186–196. 14.Choe, A., Ha, S. K., Choi, I., Choi, N., Sung, J. H. (2017). Microfluidic Gut-liver chip for reproducing the first pass metabolism. Biomedical Microdevices, 19(1). 15.Choi, H. J., Choi, D. (2013). Successful mouse hepatocyte culture with sandwich collagen gel formation. Journal of the Korean Surgical Society, 84(4), 202. 16.Choi, S. S., Diehl, A. M. (2009). Epithelial-to-mesenchymal transitions in the liver. Hepatology, 50(6), 2007–2013. 17.Civitelli, R., Fedde, K. N., Harter, J., Halstead, L. R., Gennari, C., Avioli, L. V. (1989). Effect of L-lysine on cytosolic calcium homeostasis in cultured human normal fibroblasts. Calcified Tissue International, 45(3), 193–197. 18.Clark, H. E., Mertz, E. T., Kwong, E. H., Howe, J. M., DeLong, D. C. (1957). Amino Acid Requirements of Men and Women. The Journal of Nutrition, 62(1), 71–82. 19.Combes, R. D., Balls, M. (2014). The Three Rs — Opportunities for Improving Animal Welfare and the Quality of Scientific Research. Alternatives to Laboratory Animals, 42(4), 245–259. 20.Convery, N., Gadegaard, N. (2019). 30 years of microfluidics. Micro and Nano Engineering, 2, 76–91. 21.DABEVA, M. (1993). Activation, proliferation and differentiation of liver progenitor cells into hepatocytes in rats treated with D-galactosamine. Hepatology, 18(4), A159. 22.DARVIN, P., BAEG, S. J., JOUNG, Y. H., SP, N., KANG, D. Y., BYUN, H. J., PARK, J. U., YANG, Y. M. (2015). Tannic acid inhibits the Jak2/STAT3 pathway and induces G1/S arrest and mitochondrial apoptosis in YD-38 gingival cancer cells. International Journal of Oncology, 47(3), 1111–1120. 23.Darvin, P., Joung, Y. H., Kang, D. Y., Sp, N., Byun, H. J., Hwang, T. S., Sasidharakurup, H., Lee, C. H., Cho, K. H., Park, K. D., Lee, H. K., Yang, Y. M. (2016). Tannic acid inhibits EGFR/STAT1/3 and enhances p38/STAT1 signalling axis in breast cancer cells. Journal of Cellular and Molecular Medicine, 21(4), 720–734. 24.Department of Economic and Social Affairs. (2009a). Consolidated List of Products Whose Consumption and/or Sale Have Been Banned, Withdrawn, Severely Restricted or not Approved by Governments Fourteenth Issue. 25.Dittrich, P. S., Manz, A. (2006). Lab-on-a-chip: microfluidics in drug discovery. Nature Reviews Drug Discovery, 5(3), 210–218. 26.Du, Y., Li, N., Yang, H., Luo, C., Gong, Y., Tong, C., Gao, Y., Lü, S., Long, M. (2017). Mimicking liver sinusoidal structures and functions using a 3D-configured microfluidic chip. Lab on a Chip, 17(5), 782–794. 27.Dunn, J. C., Tompkins, R. G., Yarmush, M. L. (1992). Hepatocytes in collagen sandwich: evidence for transcriptional and translational regulation. The Journal of Cell Biology, 116(4), 1043–1053. 28.Esch, E. W., Bahinski, A., Huh, D. (2015). Organs-on-chips at the frontiers of drug discovery. Nature Reviews Drug Discovery, 14(4), 248–260. 29.EvaluatePharma®. (2016). World Preview 2016, Outlook to 2022. London United Kingdom. Available at: https://www.evaluate.com/ 30.EvaluatePharma ®. (2019). World Preview 2019,Outlook to 2024. London United Kingdom. Available at: https://www.evaluate.com/ 31.Fausto, N. (2004). Liver regeneration and repair: Hepatocytes, progenitor cells, and stem cells. Hepatology, 39(6), 1477–1487. 32.Fausto, N., Campbell, J. S. (2003). The role of hepatocytes and oval cells in liver regeneration and repopulation. Mechanisms of Development, 120(1), 117–130. 33.Gerets, H. H. J., Tilmant, K., Gerin, B., Chanteux, H., Depelchin, B. O., Dhalluin, S., Atienzar, F. A. (2012). Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biology and Toxicology, 28(2), 69–87. 34.Gordon, G. J., Coleman, W. B., Hixson, D. C., Grisham, J. W. (2000). Liver Regeneration in Rats with Retrorsine-Induced Hepatocellular Injury Proceeds through a Novel Cellular Response. The American Journal of Pathology, 156(2), 607–619. 35.Guo, L., Dial, S., Shi, L., Branham, W., Liu, J., Fang, J.-L., Green, B., Deng, H., Kaput, J., Ning, B. (2010). Similarities and Differences in the Expression of Drug-Metabolizing Enzymes between Human Hepatic Cell Lines and Primary Human Hepatocytes. Drug Metabolism and Disposition, 39(3), 528–538. 36.Guo, S., DiPietro, L. A. (2010). Factors Affecting Wound Healing. Journal of Dental Research, 89(3), 219–229. 37.Haimes, J., Kelley, M. (2010). Demonstration of a ΔΔCq Calculation Method to Compute Relative Gene Expression from qPCR Data. Thermo Scientific Tech Note, 1-4. 38.Ho, C.-T., Lin, R.-Z., Chang, W.-Y., Chang, H.-Y., Liu, C.-H. (2006). Rapid heterogeneous liver-cell on-chip patterning via the enhanced field-induced dielectrophoresis trap. Lab on a Chip, 6(6), 724. 39.Ho, C.-T., Lin, R.-Z., Chen, R.-J., Chin, C.-K., Gong, S.-E., Chang, H.-Y., Peng, H.-L., Hsu, L., Yew, T.-R., Chang, S.-F., Liu, C.-H. (2013). Liver-cell patterning Lab Chip: mimicking the morphology of liver lobule tissue. Lab on a Chip, 13(18), 3578. 40.Hou, Y.-T., Ijima, H., Matsumoto, S., Kubo, T., Takei, T., Sakai, S., Kawakami, K. (2010). Effect of a hepatocyte growth factor/heparin-immobilized collagen system on albumin synthesis and spheroid formation by hepatocytes. Journal of Bioscience and Bioengineering, 110(2), 208–216. 41.Hu, Z.-Z., Huang, H., Wu, C. H., Jung, M., Dritschilo, A., Riegel, A. T., Wellstein, A. (2011). Omics-Based Molecular Target and Biomarker Identification. Methods in Molecular Biology (Clifton, N.J.), 719, 547–571. 42.Huh, D., Hamilton, G. A., Ingber, D. E. (2011). From 3D cell culture to organs-on-chips. Trends in Cell Biology, 21(12), 745–754. 43.Huh, D., Matthews, B. D., Mammoto, A., Montoya-Zavala, M., Hsin, H. Y., Ingber, D. E. (2010). Reconstituting organ-level lung functions on a chip. Science (New York, N.Y.), 328(5986), 1662–1668. 44.Huh, D., Torisawa, Y., Hamilton, G. A., Kim, H. J., Ingber, D. E. (2012). Microengineered physiological biomimicry: Organs-on-Chips. Lab on a Chip, 12(12), 2156. 45.Ikeda, S., Mitaka, T., Harada, K., Sugimoto, S., Hirata, K., Mochizuki, Y. (2002). Proliferation of rat small hepatocytes after long-term cryopreservation. Journal of Hepatology, 37(1), 7–14. 46.Kang, Y. B. A., Sodunke, T. R., Lamontagne, J., Cirillo, J., Rajiv, C., Bouchard, M. J., Noh, M. (2015). Liver sinusoid on a chip: Long-term layered co-culture of primary rat hepatocytes and endothelial cells in microfluidic platforms. Biotechnology and Bioengineering, 112(12), 2571–2582. 47.Kon, J., Ooe, H., Oshima, H., Kikkawa, Y., Mitaka, T. (2006). Expression of CD44 in rat hepatic progenitor cells. Journal of Hepatology, 45(1), 90–98. 48.Kyffin, J. A., Sharma, P., Leedale, J., Colley, H. E., Murdoch, C., Harding, A. L., Mistry, P., Webb, S. D. (2019). Characterisation of a functional rat hepatocyte spheroid model. Toxicology in Vitro, 55, 160–172. 49.Lee, P. J., Hung, P. J., Lee, L. P. (2007). An artificial liver sinusoid with a microfluidic endothelial-like barrier for primary hepatocyte culture. Biotechnology and Bioengineering, 97(5), 1340–1346. 50.Lemire, J. M., Shiojiri, N., Fausto, N. (1991). Oval cell proliferation and the origin of small hepatocytes in liver injury induced by D-galactosamine. American Journal of Pathology, 139(No. 3), 535–551. 51.Lin, W.-C., Lin, W.-L. (2006). Ameliorative effect ofGanoderma lucidumon carbon tetrachloride-induced liver fibrosis in rats. World Journal of Gastroenterology, 12(2), 265. 52.Lucchetta, E. M., Lee, J. H., Fu, L. A., Patel, N. H., Ismagilov, R. F. (2005). Dynamics of Drosophila embryonic patterning network perturbed in space and time using microfluidics. Nature, 434(7037), 1134–1138. 53.Ma, L.-D., Wang, Y.-T., Wang, J.-R., Wu, J.-L., Meng, X.-S., Hu, P., Mu, X., Liang, Q.-L., Luo, G.-A. (2018). Design and fabrication of a liver-on-a-chip platform for convenient, highly efficient, and safe in situ perfusion culture of 3D hepatic spheroids. Lab on a Chip, 18(17), 2547–2562. 54.Materne, E.-M., Tonevitsky, A. G., Marx, U. (2013). Chip-based liver equivalents for toxicity testing – organotypicalness versus cost-efficient high throughput. Lab on a Chip, 13(18), 3481. 55.Mitaka, T., Kojima, T., Mizuguchi, T., Mochizuki, Y. (1995). Growth and Maturation of Small Hepatocytes Isolated from Adult Rat Liver. Biochemical and Biophysical Research Communications, 214(2), 310–317. 56.Mitaka, Toshihiro, Mikami, M., Sattler, G. L., Pitot, H. C., Mochizuki, Y. (1992). Small cell colonies appear in the primary culture of adult rat hepatocytes in the presence of nicotinamide and epidermal growth factor. Hepatology, 16(2), 440–447. 57.Mitaka, Toshihiro, Ooe, H. (2009). Characterization of hepatic-organoid cultures. Drug Metabolism Reviews, 42(3), 472–481. 58.Mitaka, Toshihiro, Sato, F., Mizuguchi, T., Yokono, T., Mochizuki, Y. (1999). Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells. Hepatology, 29(1), 111–125. 59.NAKAMURA, T., MIZUNO, S. (2010). The discovery of Hepatocyte Growth Factor (HGF) and its significance for cell biology, life sciences and clinical medicine. Proceedings of the Japan Academy, Series B, 86(6), 588–610. 60.Ooe, H., Kon, J., Miyamoto, S., Ozone, Y., Ninomiya, S., Mitaka, T. (2006). Cytochrome P450 Expression of Cultured Rat Small Hepatocytes after Long-Term Cryopreservation. Drug Metabolism and Disposition, 34(10), 1667–1671. 61.Pulavendran, S., Rose, C., Mandal, A. (2011). Hepatocyte growth factor incorporated chitosan nanoparticles augment the differentiation of stem cell into hepatocytes for the recovery of liver cirrhosis in mice. Journal of Nanobiotechnology, 9(1), 15. 62.Rajendran, D., Hussain, A., Yip, D., Parekh, A., Shrirao, A., Cho, C. H. (2017). Long-term liver-specific functions of hepatocytes in electrospun chitosan nanofiber scaffolds coated with fibronectin. Journal of Biomedical Materials Research Part A, 105(8), 2119–2128. 63.Seok, J., Warren, H. S., Cuenca, A. G., Mindrinos, M. N., Baker, H. V., Xu, W., Richards, D. R., McDonald-Smith, G. P., Gao, H., Hennessy, L., Finnerty, C. C., López, C. M., Honari, S., Moore, E. E., Minei, J. P., Cuschieri, J., Bankey, P. E., Johnson, J. L., Sperry, J., … Tompkins, R. G. (2013). Genomic responses in mouse models poorly mimic human inflammatory diseases. Proceedings of the National Academy of Sciences, 110(9), 3507–3512. 64.Shah, U.-K., Mallia, J. de O., Singh, N., Chapman, K. E., Doak, S. H., Jenkins, G. J. S. (2018). Reprint of: A three-dimensional in vitro HepG2 cells liver spheroid model for genotoxicity studies. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 834, 35–41. 65.Sinha, S., Goel, S. (2009). Effect of amino acids lysine and arginine on fracture healing in rabbits: A radiological and histomorphological analysis. Indian Journal of Orthopaedics, 43(4), 328. 66.Skardal, A., Murphy, S. V., Devarasetty, M., Mead, I., Kang, H.-W., Seol, Y.-J., Shrike Zhang, Y., Shin, S.-R., Zhao, L., Aleman, J., Hall, A. R., Shupe, T. D., Kleensang, A., Dokmeci, M. R., Jin Lee, S., Jackson, J. D., Yoo, J. J., Hartung, T., Khademhosseini, A., … Atala, A. (2017). Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform. Scientific Reports, 7(1). 67.Smriga, M., Torii, K. (2003). L-Lysine acts like a partial serotonin receptor 4 antagonist and inhibits serotonin-mediated intestinal pathologies and anxiety in rats. Proceedings of the National Academy of Sciences, 100(26), 15370–15375. 68.Soldatow, V. Y., LeCluyse, E. L., Griffith, L. G., Rusyn, I. (2013). In vitro models for liver toxicity testing. Toxicol. Res., 2(1), 23–39. 69.Tamura, T., Sakai, Y., Nakazawa, K. (2007). Two-dimensional microarray of HepG2 spheroids using collagen/polyethylene glycol micropatterned chip. Journal of Materials Science: Materials in Medicine, 19(5), 2071–2077. 70.Tompkins, R. G. (2014). SP0136 Genomic Responses in Mouse Models Poorly Mimic Human Inflammatory Diseases. Annals of the Rheumatic Diseases, 73(Suppl 2), 37.3-37. 71.Tsao, C.-W. (2016). Polymer Microfluidics: Simple, Low-Cost Fabrication Process Bridging Academic Lab Research to Commercialized Production. Micromachines, 7(12), 225. 72.U.S. Department of Health and Human Services Food and Drug Administration. (2009b). Guidance for Inustry Drug-Induced Liver Injury: Premarketing Clinical Evaluation. 73.Vig, P., Russo, F. P., Edwards, R. J., Tadrous, P. J., Wright, N. A., Thomas, H. C., Alison, M. R., Forbes, S. J. (2006). The sources of parenchymal regeneration after chronic hepatocellular liver injury in mice. Hepatology, 43(2), 316–324. 74.Whitesides, G. M. (2006). The origins and the future of microfluidics. Nature, 442(7101), 368–373. 75.Wilkening, S. (2003). COMPARISON OF PRIMARY HUMAN HEPATOCYTES AND HEPATOMA CELL LINE HEPG2 WITH REGARD TO THEIR BIOTRANSFORMATION PROPERTIES. Drug Metabolism and Disposition, 31(8), 1035–1042. 76.Yamada, M., Utoh, R., Ohashi, K., Tatsumi, K., Yamato, M., Okano, T., Seki, M. (2012). Controlled formation of heterotypic hepatic micro-organoids in anisotropic hydrogel microfibers for long-term preservation of liver-specific functions. Biomaterials, 33(33), 8304–8315. 77.Yoon No, D., Lee, K.-H., Lee, J., Lee, S.-H. (2015). 3D liver models on a microplatform: well-defined culture, engineering of liver tissue and liver-on-a-chip. Lab on a Chip, 15(19), 3822–3837. 78.Zeisberg, E. M., Tarnavski, O., Zeisberg, M., Dorfman, A. L., McMullen, J. R., Gustafsson, E., Chandraker, A., Yuan, X., Pu, W. T., Roberts, A. B., Neilson, E. G., Sayegh, M. H., Izumo, S., Kalluri, R. (2007). Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nature Medicine, 13(8), 952–961. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66939 | - |
| dc.description.abstract | 開發一套能精準模擬體內器官的體外平台一直是科學界研究的目標，這是因為在傳統的新藥試驗方法過程中，往往需要大量的動物實驗以及臨床實驗來確認藥物毒性對人體是否有害，然而這樣的方法不但需要大量研究經費與花費長期的時間，而且最終能成功通過試驗的新藥比例卻相當有限。由於微流道模型不但能夠有效模擬人體內器官結構與環境、且亦能利用高通量的檢測來縮短試驗時間、細胞更能進一步在相似體內結構的環境下生長並進行測試，這樣的做法不但提高新藥檢測效率也提升了試驗結果的可靠度，因此微流道器官晶片的研究在近年來受到很大的注目。 在體內的器官當中，最大的藥物代謝相關器官是肝臟，他同時具有合成、排泄、解毒等功能。肝臟主要由四種細胞組成：成熟肝細胞 (Primary hepatocytes) 、內皮細胞 (Liver sinusoidal endothelial cells) 、星狀細胞 (Stellate cells) 巨噬細胞 (Kupffer cells) 等，這些細胞藉由細胞與細胞之間的相互作用而組成一個具有功能的肝臟器官。本篇所研究的可拆卸式肝臟微流道晶片，在細胞來源的選擇方面使用大鼠具有分化能力的前驅幹細胞 (Liver progenitor cell) ；而在流道的選擇方面，本研究使用具有生物相容性的聚二甲基矽氧烷 (Polydimethylsiloxane，PDMS) 作為流道的材料，最後再將小型肝細胞培養於可拆卸式肝臟微流道晶片 (Detachable microfluidic liver platform) 上。將小型細胞培養於模擬體內肝臟的微流道晶片上，以此微流道晶片來當作研發新藥物的體外細胞測試平台。我們除了定期記錄培養期間的細胞型態變化之外、 亦利用酵素免疫分析 (Enzyme-linked immunosorbent assay，ELISA) 方法和定量連鎖聚合酶反應 (Quantitative real-time PCR，qPCR) 來檢測細胞白蛋白 (Albumin) 之實際分泌量及基因表現量；另外，我們也使用細胞免疫染色法來觀察細胞分化的過程 (Cytokeratin 18，成熟肝細胞標記) ，並同時檢驗肝細胞於微流道裝置中的尿素代謝之功能。 本研究結果得知：(1) 相較初代成熟肝細胞在培養 1 周後會失去細胞特性，小型肝細胞，且可以在體外環境下長時間培養至少 3 周，並且維持細胞聚落 (Colony) 型態；(2) 使用可拆卸式的PDMS 微流道來培養小型肝細胞，其小型肝細胞在第 9 天後仍能維持白蛋白分泌量，且隨著培養時間的增加其肝機能的分泌仍然保持穩定；(3) 小型肝細胞在使用注射幫浦的微流道系統以製造動態養分供給的培養環境下，會比起不使用注射幫浦的靜態微流道系統提升至少 2 倍以上的白蛋白分泌量及至少 1 倍以上的尿素代謝量。 另外一方面，於此可拆卸式肝臟微流道晶片上再加入離胺酸 (Lysine) 培養後，本研究結果亦顯示：(1) 使用含量 0.1 mg/mL 的離胺酸培養下，小型肝細胞不論在動態培養或是靜態培養下，皆可以維持 Colony 型態到第 14 天；(2) 而在培養第 3、6 天培養下的離胺酸動態白蛋白分泌量，高出離胺酸靜態培養下約 39%、34%。(3) 在培養的第 3、6、10 天，離胺靜態培養下的白蛋白分泌量比未添加離胺酸的靜態培養提升約 6倍 、19倍 、18倍。(4) 在培養的第 3、6、10 天，添加離胺酸動態培養下的白蛋白分泌量比未添加離胺酸的動態培養提升 4 倍、16 倍 、17 倍。 而於此可拆卸式肝臟微流道晶片上再另外加入單寧酸 (Tannic acid) 培養後，本研究結果證明：(1) 使用含量 1 μg/mL 的單寧酸培養下，小型肝細胞不論在動態培養或是靜態培養下，皆可以維持 Colony 型態到第 14 天；(2) 而在培養第 7、 10、 14 天培養下的單寧酸動態白蛋白分泌量，高出單寧酸靜態培養下約 3~4 倍。 此外，若在針對上述條件在培養到第 7 天後的現象看來，(1) 添加單寧酸之靜態培養下的白蛋白分泌量，會比未添加單寧酸的靜態培養提升約 4 倍以上、也比成熟肝細胞的靜態培養提升約 100 倍；(2) 添加單寧酸之動態培養下的白蛋白分泌量比未添加單寧酸的動態培養提升約 15 倍以上、 也比成熟肝細胞的靜態培養提升約 500 倍。 綜觀以上的結果，我們相信這樣的研究方向與目的對於未來肝臟晶片的建立以及長時間藥物測試的體外平台發展，能夠具有一定的參考價值和意義。 | zh_TW |
| dc.description.abstract | Developing a microfluidics platform which is efficient and time-saving for drug toxicity assessment is always a goal that scientists have been working on it. However, in vitro study of drug tests usually takes lots of time and money but put less new therapy in the market. Thus, we believe that organ-on-a-chip can be a convenient and easy way to test drug toxicity and safety in the future. The appearance of organ-on-a-chip provides a great opportunity for in vitro drug toxicity assessment without performing animal experiments. Additionally, the largest organ which has metabolism and detoxification activity in the human body is the liver, which has the function of metabolic activity, secretion of protein synthesis and energy production. Physiologically, the liver is composed of four kinds of cells – Kupffer cells, stellate cells, the liver sinusoidal endothelial cells (LSECs), and hepatocytes. Primary rat small hepatocytes are progenitor cells that have a tendency to differentiate into specific cells. In this paper, we cultured primary rat small hepatocytes on a detachable microfluidic device and used a syringe pump to mimic the blood flow condition in vivo, the prototype of liver-on-a-chip is formed. Our microfluidic platform is made of polydimethylsiloxane (PDMS) as material and can assemble easily and disassemble conveniently in order to collect cells and medium to test liver function. The PDMS microfluidic chip is also reusable, so it can lessen experiment materials and shorten the experiment time which is used to produce abandon single-used microfluidic chips. The results exhibit that (1) small hepatocytes can survive in 2D culture and form 300-400μm colonies in order to maintaining hepatocyte functions. Different from primary hepatocytes which normally maintain their morphology and function for only 7 days, small hepatocytes have great performance on both morphology and liver-specific function for at least 21 days. (2) Moreover, we analyzed the gene expression of the equipment by quantitative real-time PCR (qPCR) and the expression of albumin and Tryptophan 2,3-dioxygenase (a marker of primary hepatocytes) are increased. On the other hand, albumin secretion by enzyme-linked immunosorbent assay (ELISA) was increased after culturing 9 days. (3) Small hepatocytes which were cultured in the detachable microfluidic system which was connected to syringe pump secreted at least 2 times higher albumin and about 1 times higher urea than without syringe pump. All of these results indicated that this liver-on-a-chip had well performance in vitro culture condition and primary rat small hepatocytes performance well liver-specific function, so we thought it can be a convenience system for hepatocyte-related experiments and it can be used to drug experiments to test toxicity. Then, we choose lysine and tannic acid as drug components to test this detachable microfluidic system. First results showed that small hepatocytes cultured by 1 mg/mL lysine-contained DMEM/F12 medium represents higher albumin secretion, urea secretion and higher gene expression of albumin than cultured by DMEM/F12 medium only. (1) Small hepatocytes cultured in flow system or static system both maintained morphology of colony after culturing 14 days. (2) Albumin secretion in flow system were about 39% and 34% higher than static condition while small hepatocytes were cultured 3 and 6 days. (3) Albumin secretion of small hepatocytes cultured by lysine-contained medium in static system were about 6 times ,19 times, and 18 times higher than DMEM/F12 medium in static system after culturing 3 days, 6 days and 10 days. (4) Albumin secretion of small hepatocytes cultured by lysine-contained medium in flow system were about 4 times, 16 times and 17 times higher than by DMEM/F12 medium in flow system after culturing 3 days, 6 days and 10 days. Second results showed that small hepatocytes cultured by 0.1 μg/mL tannic acid-contained DMEM/F12 medium represents higher albumin secretion, urea secretion and higher gene expression of albumin than cultured by DMEM/F12 medium only. (1) Small hepatocytes cultured in flow system or static system both maintained morphology of colony after culturing 14 days. (2) Albumin secretion in flow system were about 3-4times higher than static condition while small hepatocytes were cultured 7, 10 and 14 days. On the other hand, when we focused on the results after small hepatocytes cultured 7 days. (1) Albumin secretion of small hepatocytes cultured in static system with tannic acid-contained DMEM/F12 medium increased about 4 times than small hepatocytes cultured in static system with DMEM/F12 medium only, and also increased about 100 times than mature hepatocytes cultured with WE medium in the static system. (2) Albumin secretion of small hepatocytes cultured in flow system with tannic acid-contained DMEM/F12 medium increased about 15 times than small hepatocytes cultured in flow system with DMEM/F12 medium only, and also increased about 500 times than mature hepatocytes cultured with WE medium in the static system. In conclusion, we hope that such research purpose provides a well value of build-up liver-on-a-chip system and in vitro long-time drug test platform in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T01:15:18Z (GMT). No. of bitstreams: 1 U0001-1608202022471100.pdf: 5568571 bytes, checksum: 9f007207ba256c80766d4d2865b33144 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 誌謝 i 摘要 ii Abstract v 目錄 viii 圖目錄 xi 表目錄 xiv 第一章 前言 1 1.1.背景 1 1.2.研究目的 3 1.3.研究架構 4 第二章 文獻探討 5 2.1.新藥發展過程 5 2.2.PDMS微流道系統應用於細胞的培養 8 2.3.器官晶片 10 2.4.肝臟晶片 13 2.5.肝前驅細胞 18 2.6.離胺酸 (Lysine) 21 2.7.單寧酸 (Tannic acid) 24 第三章 試驗設備與方法 26 3.1.實驗藥品、耗材、儀器設備與實驗動物 26 3.1.1.實驗藥品 26 3.1.2.實驗耗材 27 3.1.3.實驗儀器設備 28 3.1.4.實驗動物 29 3.2.蓋玻片培養前處理 29 3.3.肝細胞採取與培養 30 3.4.PDMS 微流道晶片製備及細胞培養 33 3.5.實驗相關檢測方法 35 3.5.1.白蛋白 (Albumin) 分泌量檢測 35 3.5.2.尿素(Urea) 活性檢測 35 3.5.3.免疫螢光染色 36 3.5.4.Quantitative real-time PCR (qPCR) 37 3.5.5.MTT細胞活性檢測 40 第四章 結果與討論 41 4.1.小型肝細胞與成熟肝細胞於體外培養之型態比較 41 4.2.PDMS微流道系統製作結果 44 4.3.PDMS微流道系統培養小型肝細胞結果 46 4.3.1.靜態培養與動態培養結果 46 4.3.2.免疫螢光染色 47 4.3.3.白蛋白分泌量 48 4.3.4.尿素分泌量 49 4.3.5.Quantitative real-time PCR (qPCR) 50 4.4.可拆式微流道使用離胺酸培養小型肝細胞之結果 52 4.4.1.靜態與動態培養的結果 52 4.4.2.白蛋白分泌量 54 4.4.3.尿素分泌量 55 4.4.4.Quantitative real-time PCR (qPCR) 57 4.5.可拆式微流道中使用單寧酸培養小型肝細胞之結果 58 4.5.1.靜態培養與動態培養結果 58 4.5.2.白蛋白分泌量 61 4.5.3.尿素分泌量 62 4.5.4.Quantitative real-time PCR(qPCR) 63 第五章 結論與未來展望 67 5.1.結論 67 5.2.未來展望 69 第六章 參考文獻 71 | |
| dc.language.iso | zh-TW | |
| dc.title | 小型肝細胞培養技術應用於可拆式肝臟晶片的開發 | zh_TW |
| dc.title | Liver-on-a-chip: Primary rat small hepatocytes in a detachable microfluidic platform
| en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 許聿翔(Yu-Hsiang Hsu),許書豪(Shu-Hao Hsu) | |
| dc.subject.keyword | 小型肝細胞,可拆卸式肝臟微流道晶片,離胺酸,單寧酸, | zh_TW |
| dc.subject.keyword | Primary rat small hepatocytes,Detachable microfluidic platform,Lysine,Tannic acid, | en |
| dc.relation.page | 80 | |
| dc.identifier.doi | 10.6342/NTU202003626 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-08-20 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 生物機電工程學系 | zh_TW |
| 顯示於系所單位： | 生物機電工程學系 | |
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