製備乳糖-蠶絲蛋白多孔性海綿支架於肝臟組織工程的應用

Abstract

肝臟疾病一直是台灣及世界其他國家的人民主要的死亡原因，無論是酒精、疾病或藥物引起的肝炎或是肝癌都會造成肝臟受損，可能導致嚴重的肝衰竭。目前臨床上比較有效治療肝衰竭的方法為肝臟移植，然而肝臟的來源一直是供不應求，並且接受肝臟移植的病患必須長期服用免疫抑制劑，因此近年來便開始發展肝臟組織工程，希望能提供更多替代治療方式，延長病患的生命。肝臟組織工程包括了肝細胞移植(hepatocyte transplantation)、組織工程移植物(tissue-engineered grafts)和人工肝臟輔助裝置(liver assist devices, LADs)。肝臟組織工程發展以來遇到最大的困難之一是肝細胞無法在體外長時間維持其活性且會快速的去分化，近年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。 多孔性材料可製成類似細胞外基質三維結構，並且具有高度的孔隙度讓養分通透及細胞貼附，因而促進細胞的貼附、增生、遷移和分化等功能。因此本實驗利用蠶絲良好的生物相容性和機械性質，結合半乳糖基，幫助肝細胞形成球狀聚集，模擬體內的三維環境；使用冷凍乾燥製備多孔性乳糖-三聚氰氯-蠶絲蛋白海綿支架，再將肝細胞培養於支架，觀察肝細胞與支架的相互作用，期望能夠延長肝細胞在體外培養的時間。 研究結果證實多孔性乳糖-三聚氰氯-蠶絲蛋白海綿支架能夠作為肝細胞培養的支架，長期培養下能維持肝細胞的存活率及功能性；此外利用非肝細胞株共同培養的方式更能增進肝細胞的功能性表現。雖然影響肝細胞功能表現的原因仍須進一步確認，但結合乳醣-蠶絲蛋白支架、肝細胞及非肝細胞株共同培養的系統應用於肝臟組織工程上是有相當的發展性。

Liver diseases have threatened people’s lives in the world. Especially in Taiwan, liver diseases are the major cause of death throughout the years. Alcohol, drugs, virus-induced hepatitis, and malignant tumor may cause liver damage and result in liver failure. In the treatment of liver failure, liver transplantation has been established as the most effective option. However, there is limitation due to the shortage of donated organ supply, and the immunosuppressive therapy must be administrated to prevent immunological rejection of transplanted tissue. In this decade, hepatic tissue engineering, which includes hepatocyte transplantation, tissue engineered grafts and liver assist devices (LADs), has been investigated in attempt to provide alternative approaches. One of the major limitation of hepatic tissue engineering is the short-term viability and rapid de-differentiation of hepatocytes in vitro. To overcome this problem, recent studies have indicated the combination of appropriate scaffold, growth factor, and co-culture with other types of cells when culturing hepatic cells. In this study, porous sponge made by silk fibroin is used to be the 3D scaffold in hepatic tissue engineering. Porous scaffold is like the 3D structure of extra-cellular matrix (ECM) in morphology and provides suitable microenvironment for higher seeding density and exchange of nutrients, oxygen, and metabolites. Silk fibroin is the biodegradable and biocompatible material, and it can provide good mechanical properties. Silk fibroin conjugated with lactose can approve the formation of spherical cell pellet to mimic 3D microstructure of hepatocyte in vivo. By observing the interaction of hepatocytes and lactose-cyanuric chloride-silk fibroin porous sponge manufactured by lyophilization, the results demonstrated that hepatocytes had good viability and normal metabolic function while culturing over a long period of time. On the other hand, hepatocytes co-cultured with the nonparenchtmal cells may approve the hepatocellular specific functions. Although the cell-cell interaction and the soluble factors between hepatocytes and nonparenchtmal cells requires further understanding, the combination of lactose-cyanuric chloride-silk fibroin porous sponge, hepocytes, and nonparechymal cells co-culturing system has the potential in hepatic tissue engineering application.