從DMN誘導肝臟纖維化的大鼠動物模型中尋找生物標記並探討肝臟纖維化的可逆機制

Abstract

此篇論文的研究目的在於探討可逆性肝臟纖維化和不可逆性肝臟纖維化在大鼠血清中蛋白質的變化，藉此剖析初期肝臟纖維化在可逆性與不可逆性之分水嶺時，調控肝臟纖維化選擇日後發展路徑的可能機制。本篇論文中，利用致癌劑 dimethylnitrosamine (DMN) 誘導大鼠產生肝臟纖維化，在纖維化不同程度以及休息不同週數的個別時期，經過肝臟切片、免疫染色確定纖維化程度，並定義肝臟纖維化可逆與不可逆的時間點。抽取肝臟纖維化不同時期的大鼠血清進行蛋白質體分析；藉由二維電泳 (Two Dimensional Electrophoresis)、膠體內水解(In gel digestion)、質譜分析，並利用PDQUEST 分析軟體，鑑定出有差異的蛋白質，其中包括血紅素結合蛋白 (haptoglobin)，脂蛋白E (Apolipoprotein E)及補體蛋白C3 (Complement C3)等蛋白質。依據鑑定出表現量有差異的蛋白質，透過生物資訊軟體MetacoreTM, 分析在統計上有顯著意義的網絡 (Network)，發現Alternative complement pathway是參與調控肝臟纖維化，選擇可逆與不可逆性路徑的可能機制。補體蛋白C3被視為是調控肝細胞增殖的重要因子，但目前文獻並沒有提到補體蛋白C3與肝臟纖維化可逆性的關係。本研究的重要結果在於，由MetacoreTM分析的網絡中發現，基質金屬蛋白酵素2,9及基質金屬蛋白酵素抑制物1,2，參與由補體蛋白C3所調控的網絡，而基質金屬蛋白酵素及基質金屬蛋白酵素抑制物是影響肝臟纖維化重要的酵素。因此，研究結果顯示，補體蛋白C3可能扮演調控可逆性與不可逆性肝臟纖維化的重要角色。

The aim of study is to compare protein expression profile between reversible and irreversible liver fibrosis in DMN-induced rat model and find out the pivotal pathway regulating the development of liver fibrosis. In this study, DMN (Dimethylnitrosamine), carcinogen, was used to induce liver fibrosis in rat. The degree of liver fibrosis was determined by histological analysis of liver sections. Immunohistochemistry stain of liver sections was performed in different liver fibrosis stages for timing definition of reversible and irreversible liver fibrosis in the DMN-induced rat model. Collecting the rat serum in different stages of fibrosis followed by proteomic analysis, which included 2DE (two-dimensional electrophoresis)、In-gel digestion and matrix-assisted laser desorption/ionization mass spectrometry. The differentially expressed protein spots were analyzed by computer-assisted image analysis (PDQEST) software (p<0.05). Identified proteins included Hp (haptoglobin), ApoE (apolipoprotein E) and complement C3. Based on the identified proteins, MetacoreTM was applied to organize network relevant to the identified proteins. The bioinformatics tool-based analyzed result shows Alternative complement pathway might be associated with the reversible regulation of liver fibrosis. Complement C3 is regard as a pivotal factor regulating the proliferation of hepatocyte. The result indicated complement C3 might be an of importance factor regulating the development of liver fibrosis, up to now, no studies elucidate the role of complement C3 in the reversibility of liver fibrosis. We also found MMPs and TIMPs involved in complement C3 regulated pathway. The functional role of MMPs and TIMPs in liver fibrosis was studied in a number of previous researches, but detail still not completely understood. Taken together, complement C3 might serve as a notable mediator involved in reversibility of liver fibrosis.