肝細胞生長因子與肝素固定化的脫細胞化肝臟間質應用於肝組織工程的研究

Abstract

為了克服器官捐贈的不足以及肝切除手術對病患所造成的負擔，本研究擬開發使受損肝臟的肝細胞恢復的肝臟貼片。肝臟貼片是利用肝組織工程(Liver tissue engineering)的脫細胞化技術所得到的肝臟脫細胞外間質(Decellularized liver ma-trix; DLM)作為本體，並將肝細胞生長因子(Hepartocyte growth factor, HGF)/肝素(Heparin)複合體修飾固定於DLM上。實驗結果得知: (1) Heparin固定於DLM film上的密度約為30 μg/cm2；(2)培養三天後，正常肝細胞在HGF/heparin-immobilized DLM的培養條件下，細胞相對生存率(Relative cell viability)提升20—30 %，肝蛋白(Albumin)分泌量可提升約20 %；(3)經過半乳糖胺(D-galactosamine)毒化處理後的肝細胞在HGF/Heparin-immobilized DLM的培養條件下，Lactate Dehydrogenase (LDH) 的活性為10 milliunits/mL，比起negative control組(毒化後的肝細胞在normal dish上繼續培養)的培養條件，損傷程度只有原本的一半，並且肝細胞機培養三天後，肝細胞能夠恢復至與無毒化組別的相同水準。本研究證明了HGF/Heparin-immobilized DLM film不但具有提升肝細胞活性、且提升肝蛋白的分泌量，另外更能夠恢復受到毒化後的肝細胞的活性並延緩肝細胞毒害的效果。這個結果對於未來製造出具備修復肝臟器官的生物貼片材料，具有十分重要的意義。

In order to overcome the shortage of organ donation and injury of patients after partial hepatectomy (PH) treatment, we aimed at developing a liver film for hepato-cytes regeneration through liver tissue engineering. In this study, a HGF/heparin-immobilized decellularized liver matrix film (HGF/heparin complex coated on DLM film) was developed for hepatocytes regeneration in liver injury. In the result, (1) the amounts of immobilized heparin on DLM film was 30 μg/cm2 when the initial heparin concentrations were 1 mg/mL. (2) The relative cell viability and albu-min synthesis of the hepatocytes on HGF/heparin-immobilized DLM film was 20-30 % and 20 % superior than on normal dish at 3 days of culture, respectively. (3) The lactate dehydrogenase activity of the D-galactosamine-induced injury of hepatocytes on hepa-rin-immobilized DLM film cultures was 10 milliunits/mL, which was 50 % lower than that in the D-galactosamine-induced injury of hepatocytes on normal dish cultures, and albumin synthesis can recover to the same level as non-toxic hepatocytes cultured on normal dish. The HGF/heparin-immobilized DLM film showed highly potential in maintaining hepatocyte culture and also in repairing injured hepatocytes from D-galactosamine. It is believed that this HGF/heparin-immobilized DLM film has promising potential for hepatocyte transplantation, and could be applied for future use in liver tissue engineering.