摘要:肝臟再生是肝細胞週期被啟動,此一過程牽涉於DNA 的合成和基因的複製,但涉入肝臟再生啟動,演進及結束的主要因子仍未知。近年來肝臟移植成為治療末期肝臟疾病的最佳選擇。然而肝臟移植卻因為宿主來源不足、價格昂貴以及終生服用抗排斥藥物而限制其應用性。肝細胞移植被證明可以取代肝臟移植來治療許多肝臟疾病。過去我們的研究成果也發展出適合肝細胞移植的抗排斥藥物(發表在Hepatology 2006)及移植肝細胞可大範圍取代肝臟的宿主前治療模式(發表在Hepatology2008)。然而肝細胞移植的發展仍舊需要克服肝細胞來源短缺與異體排斥的問題。因此幹細胞移植是近年來被應用在許多疾病上並且具有其治療的可行性。幹細胞移植有可能成為協助治療肝臟疾病的新選擇。許多研究都證實間葉系幹細胞能夠做為細胞移植的來源並應用於許多肝臟疾病的治療,它是一個可以解決肝細胞來源短缺與異體排斥問題。我們先前的研究也證實間葉幹細胞具有免疫調節與旁分泌的治療潛能。近年來誘導性多功能幹細胞 (Induced pluripotent stem cells -- iPS cells)是另一個被證實能做為細胞移植的來源。在此計畫中我們將探討誘導性多功能幹細胞在纖維化肝臟中其治療效果並且著重於誘導性幹細胞與肝臟星狀細胞間的相互作用。本計畫擬分三年進行,將誘導性多能幹細胞分化成肝細胞,並送入肝纖維化的小鼠動物模式,進而評估其治療療效並鑑定其與肝臟新生之相關性。第一年: 將分成兩部分進行為小鼠活體肝細胞與肝臟星狀細胞的取得以及體外培育誘導性多能幹細胞分化成肝細胞:(1) 建立肝細胞與肝臟星狀細胞的分離與體外培養的系統。(2) 利用流式細胞儀與免疫螢光染色鑑定其肝臟星狀細胞之特性。(3) 建立誘導性多能幹細胞分化成肝細胞的系統。以體外培育方式誘導其誘導性多能幹細胞逐步走向肝細胞,並建立其分化步驟。(4) 鑑定來自誘導性多能幹細胞的肝細胞其是否具有肝細胞應有其功能性。以RT-PCR 技術檢測其分化後的肝細胞之基因表現狀態,以免疫螢光染色證實此分化後的肝細胞其蛋白質表現狀態。第二年: 探討肝細胞與肝臟星狀細胞的交互作用以及建立肝纖維化的小鼠動物模式:(1) 探討肝細胞與肝臟星狀細胞之間的交互作用。(2) 比較自然的肝細胞與誘導性多能幹細胞分化後的肝細胞,此兩種肝細胞的再生能力探討。(3) 建立肝纖維化的小鼠動物模式。以肌肉注射四氯化碳誘導小鼠的肝纖維化動物模式。(4) 評定肝纖維化程度。以免疫組織化學染色技術評定肝肝纖維化程度;以RT-PCR 技術檢測與肝纖維化相關的基因是否被啟動;以生化分析GOT、GPT、T-bill 值,藉以評定肝纖維化程度。第三年:誘導性多能幹細胞分化後肝細胞在肝纖維化的小鼠動物模式中其免疫調節能力探討:(1) 研究來自誘導性多能幹細胞分化後肝細胞是否能改善肝纖維化的程度。以第二年所建立的評定肝纖維化程度的相關實驗來評定此細胞是否能夠改善肝纖維化的程度。(2) 研究來自誘導性多能幹細胞分化後肝細胞是否能促進肝臟再生。細胞移植後,利用肝臟組織分析與肝臟再生相關的基因表現狀態。(3) 證實來自誘導性多能幹細胞分化後肝細胞是否存在於纖維化肝臟。先行標定螢光物於肝細胞並以觀察螢光來追蹤其是否存在於纖維化肝臟。從此計劃中我們不僅可了解此種新的治療方式對纖維化肝臟疾病的療效以做為將來臨床運用推廣的基礎,並可擴展將來臨床上新的移殖細胞來源。
Abstract: Liver regeneration is well documented that gene duplication and DNA synthesis occur in thecell cycle of regenerating hepatocytes. However, the key genes and their pathway for initiation,differentiation and termination of liver regeneration are behind well-known. In recent years, livertransplantation has become one of the best choices for treatment of end stage liver disease.However, liver transplantation is still limited due to donor shortage, high cost and need ofimmunosuppressive medicine. One the other way, hepatocytes transplantation has been alternativeto liver organ transplantation in many different kinds of liver diseases. Cell transplantation canreplace the whole organ transplantation as the first therapeutic choice for patient with organ failure.We demonstrated previously the suitable immunosuppression protocol for hepatocytestransplantation (published in Hepatology 2006) and the extensive liver repopulation bytransplanted hepatocytes after suitable preconditioning treatment of recipients (published inHepatology 2008). However the development of hepatocyte transplantation still needs toovercome the shortage of sources of hepatocyte and allograft rejection. Mesenchymal stem cells(MSCs) have been shown to have the therapeutic potential in several different kinds of diseasesand can overcome the problems of organ shortage and allograft rejection. Besides the ability ofdifferentiation into different cell types, the MSCs have been demonstrated to have theimmunomodulation and paracrine effects to regulate the function of neighboring cells. We foundin the preliminary studies that the MSCCM (MSC-derived condition medium) have dual beneficialfactors contributed to the regeneration of liver fibrosis in our preliminary results, which areincreased proliferation of hepatocyte and suppressed proliferation of stellate cells. Inducedpluripotent stem cells (iPS cells) have been shown to be a alternative useful source of MSCs. Weplan to study in this project whether the iPS cells immunomodulate the regeneration of liverfibrosis. We will apply proteinomics assay to characterize how the iPS cells immunomodulate theregeneration of liver fibrosis, especially focusing on the crosstalk between the iPS cells andhepatocytes and stellate cells. We plan to identify the potential molecular involved in thisimmunomodulation, focusing on the growth factors, cytokines and chemokines. And finally wewill study the therapeutic efficiency of iPS cells on the regeneration of liver fibrosis in mice model.The results of this study will help us to develop the protocol of applying iPS cells as a newtherapeutic strategy for patients with liver fibrosis. The three-year project to be carried out in thisproject. The iPS-Hep cells will transplantated in fibrosis liver, and to assess the therapeuticefficacy and correlation with the liver regeneration:The 1st Year Protocol: Obtains the primary hepatocytes, hepatic stellate cells (HSC), and toguide the iPS cells differentiation to the hepatocytes.(1) To set up the isolation system for primary hepatocyte and HSC cells.(2) Characterization of HSC by flow cytometry.(3) To set up the protocol for iPS cells differentiation into hepatocytes. Using the grower factor toguide the iPS cell to hepatocytes in vitro.(4) Characterization of functional hepatocytes. To assess the liver-relate gene expression byRT-PCR; To acquire the protein expression by immunofluorescent.The 2nd Year Protocol: Explore the cross-talk between hepatocytes and HSCs andestablishment of mice model with liver fibrosis and methodology.(1) To understand the cross-talk of hepatocytes and HSC cells.(2) Compare with the primary hepatocytes and iPS-Hep cells on regenerative ability.(3) To set up the fibrosis animal model. Using the CCl4 to induce the liver fibrosis in mice(4) To assess the degree of liver fibrosis. The degree of liver fibrosis could be measure byimmunohistology, the RT-PCR to know the fibrosis-relate gene expression, and the value ofGOT, GPT, and t-bill could be assess the damage state in the liver.The 3rd Year Protocol: The immunomodulation effect of iPS-Hep cells on the regeneration offibrotic liver.(1) To investigate whether iPS-Hep cells will be improve the severity of liver fibrosis. Base onthe 2nd results, we are going to assess the degree of fibrosis in liver(2) To study whether iPS-Hep cells perturbed the expression of fibrogenetic gene in the fibroticliver. After iPS-Hep cell transplantated, the regeneration-relate gene expression will detect byRT-PCR.(3) Traces the iPS-Hep cells localization in fibrosis liver. iPS-Hep will stain with tracking dye totrace the localization in fibrosis liver.Furthermore, we will understand the therapeutic potential of iPS-Hep in fibrosis liver disease, andmay expend in the future clinical source of novel source for cell-base therapy.