含脫細胞化肝臟間質之自癒合水膠應用於肝纖維化之恢復

Abstract

肝臟是以代謝功能為主的器官，主要有去除體內毒素、肝醣儲存、碳水化合物代謝、脂質代謝及蛋白質合成等重要功能。然而肝臟受損發炎後病變所致的慢性肝病及肝硬化成為人類常見死因之一，這些發炎症狀刺激肝臟分泌細胞外間質 (Extracellular matrix, ECM) 並積累形成疤痕組織 (Scar tissue) 降低肝功能，此過程即為肝纖維化 (Liver fibrosis)。 本研究承接實驗室過往研究，使用 CPDP 自癒合水膠 [由 Chitosan-pheol (CP), Difunctionalized PEG (DP) 所合成之具自癒合特性之材料] 作為仿生微型肝組織之載體。雖然本實驗室既往的研究中已經證實了 CPDP 自癒合水膠有以下的特性：(1) 優良的自癒合能力及可注射性；(2) 大約 0.5 kPa 且與肝臟相仿的力學強度；(3) 長達兩週以上的穩定降解時間。然而若是將 CPDP 自癒合水膠與肝細胞一同植入體內時，反而會引發免疫反應，使受損組織對外來細胞產生排斥等不良反應。因此，本研究的第一部分擬將 CPDP 自癒合水膠結合脫細胞化肝臟間質 (Decellularized Liver Matrix, DLM) 來改善載體之微環境。這是因為 DLM 是被廣泛運用且具與肝臟高度生物相容性的自體性物質，因此我們擬進行 DLM 成分及濃度最佳化的檢測，以利後續將其與 CPDP 自癒合水膠混合使用。 本研究的第二部分我們擬改善過往研究中肝細胞球 (Hepatocyte spheroids) 的製備方式，並結合人類臍靜脈內皮細胞 (Human umbilical vein endothelial cells, HUVECs) 形成包覆 HUVECs 的肝細胞球 (HUVECs-covered hepatocyte spheroids) 以製備出微型仿生肝臟組織。最後，將以四氯化碳 (Carbon Tetrachloride, CCl4) 作為誘導毒化物以建立體外肝纖維化模型，並結合 CPDP 水膠、DLM、肝細胞球及 HUVECs-covered 肝細胞球，形成 CPDP-Spheroids (CS), CPDP/DLM-Spheroids (CDS), CPDP/DLM-Spheroids and HUVECs (CDSH) 等組別之微型仿生肝臟組織，並檢測其應用於肝纖維化的恢復。期許 CPDP 自癒合水膠於肝臟組織工程中的應用能有進一步的發展、且能為肝病治療給予新的方法與展望。

The liver is an organ whose main metabolic function is to remove toxins from the body, store glycogen, carbohydrate metabolism, lipid metabolism, and protein synthesis. However, chronic liver disease and liver cirrhosis caused by liver damage and inflammation have become one of the common causes of death in humans. These inflammatory symptoms stimulate the liver to secrete extracellular matrix (ECM) and accumulate to form scar tissue, which reduces liver functions, this process is liver fibrosis. This study follows the previous research of the laboratory, using CPDP self-healing hydrogel [a material with self-healing properties synthesized from Chitosan-pheol (CP), Difunctionalized PEG (DP)] as the carrier of bionic micro-liver tissue. Although previous studies in our laboratory have confirmed that CPDP self-healing hydrogel has the following characteristics: (1) excellent self-healing ability and injectability; (2) mechanical strength of about 0.5 kPa and similar to that of liver; (3) ) stable degradation time of more than two weeks. However, if the CPDP self-healing hydrogel is implanted into the body together with liver cells, it will trigger an immune response, causing the damaged tissue to produce adverse reactions such as rejection of foreign cells. Therefore, the first part of this study intends to combine CPDP self-healing hydrogel with decellularized liver matrix (DLM) to improve the microenvironment of the carrier. This is because DLM is an autologous substance that is widely used and highly biocompatible with the liver. Therefore, we plan to optimize the composition and concentration of DLM so that it can be mixed with CPDP self-healing water gel in the future. Therefore, the first part intends to improve the microenvironment of the carrier by combining the decellularized liver matrix (DLM), which is a widely used autologous substance with high biocompatibility with the liver. For this reason, we conduct the detection of DLM composition and concentration optimization for subsequent mixed use. In the second part of this study, we intend to improve the preparation method of hepatocyte spheroids in previous studies, and combine Human umbilical vein endothelial cells (HUVECs) to form hepatocyte spheroids (HUVECs- covered hepatocyte spheroids) to prepare micro-bionic liver tissue. Finally, carbon tetrachloride (CCl4) will be used as an inducing toxicant to establish an in vitro liver fibrosis model, and combined with CPDP hydrogel, DLM, hepatocyte spheres and HUVECs-covered hepatocyte spheres to form CPDP-Spheroids (CS), CPDP/DLM-Spheroids (CDS), CPDP/DLM-Spheroids and HUVECs (CDSH) and other groups of micro-bionic liver tissues, and tested their application in the recovery of liver fibrosis. It is hoped that the application of CPDP self-healing hydrogel in liver tissue engineering can be further developed, and it can provide new methods and prospects for the treatment of liver diseases.