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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃義侑(Yi-You Huang) | |
dc.contributor.author | Peng-Lin Yeh | en |
dc.contributor.author | 葉芃伶 | zh_TW |
dc.date.accessioned | 2021-06-15T03:52:25Z | - |
dc.date.available | 2013-07-30 | |
dc.date.copyright | 2010-07-30 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-07-08 | |
dc.identifier.citation | [1]United Network for Organ Sharing. http://www.unos.orgydatay. (2009).
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44657 | - |
dc.description.abstract | 一直以來肝臟疾病對於台灣境內及許多其他國家的人民都構成莫大的生命威脅,無論是酒精、藥物、病毒引起的肝炎或是肝癌等等都會造成肝臟受損,當肝臟功能喪失時可能會導致嚴重的肝衰竭。在臨床上相對來說較有效的肝衰竭治療方法為肝臟移植,例如急性肝衰竭(acute liver failure; ALF)的患者若無法及時進行肝臟移植手術,致死率可能高達百分之八十;然而肝臟來源一直都是供不應求的,因此近幾十年來便開始發展肝臟組織工程希望能提供更多替代方式,其中包括了肝細胞移植(hepatocyte transplantation)、組織工程移植物(tissue-engineered grafts)和人工肝輔助裝置(liver assist devices, LADs)。肝臟組織工程自發展以來遇到最大的難題之一是肝細胞在體外無法長時間維持其活性且會快速的去分化(de-differentiation),近來的研究指出選擇適當的支架、加入特定生長因子和以非肝細胞株共同培養之方式可望改善此問題。
本實驗利用氣體發泡(gas foaming)法結合雙重乳化(double emulsion)法製備出具開放性孔洞之聚乳酸-甘醇酸微球體當作支架,微球體平均大小約為430μm,球體表面孔洞則集中在10-20μm。與一般使用實心微球體作為培養支架相比,肝細胞培養於具開放性孔洞的微球體可提高其細胞承載量,且能夠促進養份、氧氣和代謝物的交換。此外其具備可注射性的優點,解決了一般具孔洞性支架必須以開放性手術方式植入生物體內的問題。為了延長肝細胞在體外的功能性表現,實驗中利用纖維母細胞、間葉系幹細胞和人類臍靜脈內皮細胞分別與肝細胞進行共同培養,並以肝細胞產生的白蛋白及尿素濃度來評估其功能性;結果顯示非肝細胞株可能藉由細胞-細胞接觸的方式或者分泌特定的生物因子使得肝細胞在培養期間能夠維持其在體外的功能性。 研究結果證實,具開放性孔洞之聚乳酸-甘醇酸微球體能夠做為肝細胞培養的支架;此外利用非肝細胞共同培養的系統可延長肝細胞在體外的功能性表現長達兩週。雖然影響的原因是由細胞接觸或生物因子所導致仍須進一步確認,但結合具開放性孔洞之微球體、肝細胞及非肝細胞共同培養的系統在應用於肝臟組織工程上是有潛力的。 | zh_TW |
dc.description.abstract | Each year liver diseases have threatened people’s lives in Taiwan and many other countries. Alcohol, drug, virus-induced hepatitis, and malignant tumor can cause liver damage and result in liver failure. In the treatment of liver failure, liver transplantation has been established as an effective final option. For instance, the mortality rate of patients who suffered from acute liver failure (ALF) was reported as high as 80% without liver transplantation. However, due to the shortage of donated organ supply, hepatic tissue engineering, which includes hepatocyte transplantation, tissue-engineered grafts and liver assist devices (LADs), has been investigated for several decades in attempt to provide alternative approaches. One of the major obstacles of hepatic tissue engineering was the short-term viability and rapid de-differentiation of hepatocytes in vitro. Recent studies have indicated that combination of appropriate scaffold, factors, and co-culturing with other cell types may overcome this problem.
In this study, open porous poly (lactic-co-glycolic acid) (PLGA) microspheres were fabricated using gas foaming in a W1/O/W2 double emulsion method, with a mean size of approximately 430μm and an average pore diameter mostly between 10-20μm. Comparing with nonporous microspheres as scaffold, culturing hepatocytes with open porous microspheres provided suitable microenvironments for higher cell seeding density and exchange of nutrients, oxygen, and metabolites. Unlike porous scaffolds requiring open surgery process for implantation in the body, open porous microspheres can deliver cells with a syringe in an injectable manner, which is more patient-friendly. For further improvement in viability and specific hepatic functions, nonparenchymal cells (rat fibroblasts, rat mesenchymal stem cells and human umbilical venous endothelial cells) were each co-cultured with mature hepatocytes. Quantification of albumin secretion and urea synthesis showed that nonparenchymal cells may support hepatocellular functions due to cell-cell interactions or secretion of soluble factors. This study suggested that open porous PLGA microspheres were successfully developed and used as the hepatocytes culturing scaffold. Moreover, the hepatocellular specific functions sustained up to 2 weeks in the support of co-culturing with nonparenchymal cells. Although the cell-cell interactions and soluble factors between hepatocytes and nonparenchymal cells requires further understanding, the combination of open porous microspheres, hepatocyes, and nonparenchymal cells co-culturing system has the potential in hepatic tissue engineering application. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T03:52:25Z (GMT). No. of bitstreams: 1 ntu-99-R97548026-1.pdf: 3679818 bytes, checksum: 09430ad030fa9e0330ec195e9858ad2b (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 致謝 ………………………………………………………………………………………………………… Ι
摘要 …………………………………………………………………………………………………………………… II Abstract …………………………………………………………………………………………………… Ⅳ 圖目錄 …………………………………………………………………………………………………………………… Ⅹ 第一章 序論 ………………………………………………………………………… 1 1.1 肝臟組織工程 ………………………………………………………… 1 1.2 肝臟 ……………………………………………………………………………………………… 2 1.2.1 肝實質細胞 ……………………………………………………………………… 2 1.2.2 非肝實質細胞 …………………………………………………………………………………… 5 1.3 支架 ……………………………………………………………………………………………………………………… 7 1.4 生長因子 ……………………………………………………………………………………………………… 8 1.5 非肝細胞株對於肝細胞之調控 …………………………………………………………………………… 9 1.5.1 人類臍靜脈內皮細胞 ……………………………………………………… 9 1.5.2 間葉系幹細胞 ………………………………………………………………………………………… 10 1.5.3 NIH/3T3細胞 ……………………………………………………………………………………… 11 1.6 聚乳酸-甘醇酸 ……………………………………………………………………………………………… 11 1.6.1 聚乳酸-甘醇酸支架 ………………………………………………………………………………… 13 1.6.2 具開放性孔洞之聚乳酸-甘醇酸微球體 ………………………………………………………… 14 第二章 研究動機與目的 ……………………………………………………………………………… 16 第三章 實驗材料與方法 ……………………………………………………………………………………… 18 3.1 實驗藥品 ………………………………………………………………………………………………………… 18 3.2 實驗儀器 ………………………………………………………………………………………… 20 3.3 肝細胞初代培養 .……………………………………………………………………………………… 21 3.4 人類臍靜脈內皮細胞初代培養 ……………………………………………………………………… 28 3.5 大鼠間葉系幹細胞初代培養 ……………………………………………………… 29 3.6 NIH/3T3繼代培養 ……………………………………………………………… 29 3.7 製備具開放性孔洞之聚乳酸-甘醇酸微球體 ………………………………………………… 29 3.8 製備實心聚乳酸-甘醇酸微球體 …………………………………………………………………………… 30 3.9 掃描式電子顯微鏡觀測 …………………………………………………………… 32 3.10 光學顯微鏡觀察具孔洞之聚乳酸-甘醇酸微球體的截面 ……………………………… 32 3.11 肝細胞培養於聚乳酸-甘醇酸微球體 ……………………………………………………… 32 3.12 肝細胞與非肝細胞株共同培養於聚乳酸-甘醇酸微球體 ……………………………… 33 3.13 Hoechst 33342染色觀察細胞核 ……………………………………………………… 34 3.14 LIVE/DEADR Viability/Cytotoxicity kit染色觀察細胞活性 ……34 3.15 雙光子顯微鏡觀察肝細胞生長於具開放性孔洞之微球體的情形 ……………… 35 3.16 檢測肝細胞之功能性表現 ………………………………………………………… 35 3.16.1 白蛋白定量分析 …………………………………………………………… 35 3.16.2 尿素定量分析 …………………………………………………………………… 35 3.17 以細胞追蹤染劑標定細胞並使用共軛焦顯微鏡觀察細胞生長於具開放性孔洞之微球體的情形 ………………………………………………………… 36 3.18 掃描式電子顯微鏡觀察細胞於具開放性孔洞之微球體表面的形態………………37 第四章 結果與討論 ………………………………………………………………………………………………………38 4.1 肝細胞培養於第一型膠原蛋白塗覆之培養盤的觀察 ……………………………………………… 38 4.2 以多重乳化法製備聚乳酸-甘醇酸微球體 …………………………………………………………… 42 4.3 肝細胞培養於聚乳酸-甘醇酸微球體 …………………………………………………………………… 47 4.4 以雙光子顯微鏡觀察肝細胞於具開放性孔洞之聚乳酸-甘醇酸微球體上的分布情形 ………………………………………………………… 51 4.5以細胞追蹤染劑標定細胞並使用共軛焦顯微鏡觀察肝細胞與非肝細胞株共同培養於具開放性孔洞之微球體的分布情形 …………………………… 53 4.6 檢測白蛋白及尿素合成量以評估肝細胞培養於具開放性孔洞之微球體的特定功能性表現並探討非肝細胞株對於肝細胞之功能性表現的影響 ………………………………… 56 4.6.1 肝細胞尿素合成量之評估 …………………………………………………………………… 56 4.6.2 肝細胞白蛋白分泌量之評估 ………………………………………… 59 4.7以掃描式電子顯微鏡觀察細胞於具開放性孔洞之微球體表面的形態並探討其現象所造成的影響 ……………………………………………………………… 62 第五章 結論 ……………………………………………………………………………………… 64 參考文獻 …………………………………………………………………………………… 65 | |
dc.language.iso | zh-TW | |
dc.title | 具開放性孔洞之微球體於肝臟組織培養的應用 | zh_TW |
dc.title | Application of Open Porous Microspheres in Hepatic Tissue Cultivation | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳惠玲,黃意真 | |
dc.subject.keyword | 肝臟組織工程,溶劑鑄造/鹽析法,聚乳酸-甘醇酸微球體,肝細胞,白蛋白分泌,尿素合成, | zh_TW |
dc.subject.keyword | Hepatic tissue engineering,Gas foaming,PLGA microspheres,Hepatocytes,Albumin secretion,Urea synthesis, | en |
dc.relation.page | 69 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-07-08 | |
dc.contributor.author-college | 工學院 | zh_TW |
dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
顯示於系所單位: | 醫學工程學研究所 |
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ntu-99-1.pdf 目前未授權公開取用 | 3.59 MB | Adobe PDF |
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