肝細胞癌 (hepatocellular carcinoma, HCC) 在台灣為常見之惡性腫瘤，目前已知造成肝癌的危險因子有肝硬化、酗酒、藥物濫用、黃麴毒素、非酒精性的脂肪性肝炎、慢性病毒性肝炎等。全球有超過75%的肝癌病患成因是慢性病毒性肝炎。目前已發現肝癌衍生生長因子 (Hepatoma-derived growth factor, HDGF）在肝細胞癌化過程中扮演著重要角色，具有刺激纖維母細胞，內皮細胞和肝癌細胞生長的能力，而且HDGF 過度表現與人類肝癌，肺癌和黑色素癌之癌化過程也有密切關聯，近來有許多研究探討HDGF的表現和不同癌症的相關性。 C-反應蛋白 (C-Reactive Protein, CRP) 屬於一群獨特的血清蛋白家族，它主要是由肝臟分泌，在外傷、局部缺血、燒傷、各種發炎與感染時會迅速上升至1000倍，CRP是個急性期的蛋白質，因此檢驗 CRP可用來監測各種發炎狀況，同時也是心血管疾病的危險因子。然而，C反應蛋白在肝癌發生中的作用尚不清楚。本研究發現CRP在不同分化程度的人類肝癌細胞株均有表現。 外加CRP蛋白質能促進肝癌細胞之增生、遷移、侵襲與群落形成能力。 CRP除了可以調升CRP與受體CD64表現量外。 CRP也可調升HDGF與其受體Nucleolin的表現。 而在西方墨點分析發現CRP蛋白質能促進Akt與NFB 訊息途徑活性， 由於HDGF在肝細胞癌化與Akt/NFκB訊息途徑之功能已確認， 因此CRP在活化HDGF/Nucleolin訊息在癌化機制中可能扮演重要的角色。

Hepatocellular carcinoma (HCC) is one of the most common malignancy and known as country disease in Taiwan. Hepatoma-derived growth factor (HDGF), a mitogen for various types of cells, plays an important role in liver carcinogenesis. HDGF overexpression is associated with tumor progression and poor survival outcome in many types of cancer including lung cancer, melanoma and HCC. C-reactive protein (CRP) is a acute phase protein, belongs to a group of unique serum protein family and is mainly secreted by the liver. The serum CRP level rises rapidly by up to 1,000 times during acute or chronic inflammatory conditions such as infection and cardiovascular diseases. However, the role of CRP in liver carcinogenesis remains unclear. By using a panel of hepatoma cell lines, immunoblot analysis showed that CRP is highly expressed in all hepatoma cells regardless of their differentiation status. Exogenous CRP significantly stimulated the proliferation, wound healing, invasion and colonies formation of hepatoma cells. Besides, CRP increased CRP and its receptor CD64 expression in hepatoma cells. Interestingly, CRP also enhanced the expression of HDGF/nucleolin axis in hepatoma cells. Finally, CRP enhanced Akt phosphorylation and the expression of NFκB members. Because the role of HDGF in liver carcinogenesis and Akt/NFκB activation has been recently established, it seems plausible that CRP may mediate the malignant transformation of hepatoma cells through activation of HDGF/nucleolin axis.

論文審定書………………………………………………………………………………i
致謝………………………………………………………………………...……………ii
中文摘要 .................................................................................................iii
Abstract.....................................................................................................iv
第一章緒論Introduction................................................................................1
1.1　肝癌（Hepatoma carcinoma）.............................................................1
1.2　肝癌衍生生長因子（Hepatoma-derived growth factor, HDGF）........ 5
1.3　C-反應蛋白（C-Reactive Protein, CRP）...................................... 9
1.4 研究目的……………………………………………………………….....….....11
2.1　細胞培養液......................................................................................12
2.2　細胞培養.........................................................................................12
2.3　細胞數目之測定...............................................................................13
2.4　 Antibody 及Protein……………………………………….....…….………..13
2.5　西方墨點法 （Ｗestern blotting analysis）........................................14
2.6　細胞增殖試驗（Cell proliferation assay）.........................................15
2.7　免疫螢光染色（Immunoflurorescent Stain）.....................................15
2.8　細胞移行分析（Cell invasion assay） ............................................16
2.9　細胞群落形成（Colony formation assay）.......................................16
2.10 傷口癒合分析（Wound healing assay）..........................................16
2.11　RNA萃取（RNA Extraction）.......................................................17
2.12　qRT-PCR ..................................................................................17
2.13　免疫分析儀………………………………………………………………. 18
第三章結果Results..............................................................................19
3.1 CRP和HDGF在不同人類肝癌細胞株的表現量 .................................19
3.2添加外源性HDGF和CRP後，在Huh7細胞培養液中CRP及HDGF的
表現量……………………..…………………………………….......……… 19
3.3探討在HCC cell lines外源性的CRP和HDGF之間的關聯性…………….19
3.4 探討CRP及HDGF在Akt/NFκB pathway的關聯 ................................20
3.5 在CRP及HDGF同時存在時加劇癌化的能力.....................................21
第四章討論Discussion.......................................................................23
圖表……………………………………………………………………………..26
附錄……………………………………………………………………..………46
References......................................................................................59

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