|Abstract: ||摘要:新型藥物在活體中經由肝臟活化作用而轉化成對肝組織具有危害的有毒物質，這牽涉到肝細胞對藥物的吸收、生物轉換以及移除等作用。利用動物實驗來驗證藥物的安全性是十分耗時的；而相對於動物實驗而言，使用體外細胞培養系統可節省人力與物力的消耗。此外，體外細胞培養系統亦可用來預測藥物在活體內的代謝及藥物在生物轉換過程中所扮演的角色為誘導劑或抑制劑。目前體外肝組織系統可分為初代肝細胞、微粒體、肝臟切片、細胞株等，其中初代肝細胞被廣泛應用在藥物毒性測試。 由於肝細胞為一貼附型之細胞，而且在體外會快速喪失其肝細胞之細胞型態。所以許多的文獻藉由調控細胞培養條件來維持肝細胞的生理功能。這些方法包括將肝細胞培養在基底膜、或培養在collagen gel sandwiches、或是培養於三維結構之基質中、或者將肝細胞與其他肝組織細胞或非肝組織細胞共培養。許多研究顯示肝細胞培養在三維結構之基質中，能有效地維持肝細胞原有之細胞型態與細胞活性及功能，而改善初代肝細胞在二維平板基質中快速喪失細胞原有之型態及功能的現象。Collagen sandwiches, MatrigelTM或是利用褐藻酸微膠囊包覆肝細胞為目前常見的肝細胞培養之三維結構基質。 雖然利用褐藻酸微膠囊包覆肝細胞是常見之方法，但是此方法並無法維持肝細胞所有的細胞生理功能。根據過去我們的研究發現褐藻酸的存在會影響膠原蛋白之在再重組之特性，造成膠原蛋白D-banding特徵結構的改變。此外，亦發現此系統對初代骨母細胞的生長與分化有抑制之現象。所以由以上之結果可知褐藻酸並不是微膠囊包覆肝細胞系統中最佳的材料。 許多的文獻亦指出三維結構基質之物性與化性均會影響到肝細胞的生理功能。所以此研究計畫之目的在開發一新型核殼微膠囊包覆系統，以改善褐藻酸微膠囊包覆肝細胞之缺點。另外一方面，由於半乳糖可與肝細胞的細胞膜表面上之asialoglycoprotein receptor相結合，而使肝細胞易於維持肝細胞之原有細胞型態。因此，在第二年我們將把膠原蛋白修飾為半乳糖化之膠原蛋白，以提高肝細胞之生理功能。我們預期此系統可改善傳統微膠囊之缺點，而可用來做為長時間體外肝細胞評估藥物代謝與藥物毒性之檢測模式。|
Abstract:The capacity of a new drug to produce liver damage in vivo results from a serial of complex cellular process that are involved in the uptake, biotransformation , and elimination of potentially toxic compounds. It is time-consuming in animal studies for drug registration. In contrast with animal studies, in vitro cell culture methods are characterized as a shorter period and a less amount of compound requirement. There are several kinds of the in vitro liver systems including isolated and cultured hepatocytes, microsomes, tissue slice, immortalized cell line. The most popular system is hepatocytes cultured model, and widely used for toxicity studied. Hepatocytes are anchorage-dependent cells, and rapidly lose their hepatic phenotype in vitro. There are many methods to maintain hepatocytes functions in vitro by adjusting different culture configurations. These methods include culturing on or in basement membrane, culturing in collagen gel sandwiches, co-culturing with other liver-derived or non-liver cell types, or culturing on three dimensional matrices. Some authors proved two-dimensional culture of hepatocytes may obstruct some normal regulatory processes, and the functions of hepatocytes are better maintained in three dimensional than in two dimensional culture system. Generally, three-dimensional culturing systems include collagen sandwiches, MatrigelTM culture and hepatocytes microencapsulated in alginate. There are many advantages to support alginate microencapsulation is a good method to cultured hepatocytes. But until now, it has not yet been optimized for maintaining all of important functions of hepatocyes at desired levels in alginate encapsulation system. According our past experiments, we found that collagen fibrils exhibit a spindle shape when reconstituted in the presence of alginate. It means that extracellular matrix architecture maybe influenced by the presence of alginate. Moreover, the primary osteoblasts, anchorage-dependent cells like primary hepatocytes, entrapped in alginate will lost their viability. Together these results, we conclude that alginate may not the best choice to encapsulate hepatocytes. The aim of this project is to develop a novel core-shell microencapsulation technique that could overcome the major disadvantages in alginate system. Besides, galactose could interact with the asialogycoprotein receptor on hepatocytes to promote their functional maintenance. Then at 2nd year, we will incorporate galactose onto collagen to maintain biological functions of hepatocytes. We predict this novel microencapsulation technique would be better than traditional alginate system. Finally, we could utilize this system to establish an in vitro liver cell model for long-term toxicology and drug metabolism testing.