惡性的黑色素癌在人類的癌症中是發展最為快速的惡性腫瘤，尤其好發於年輕人肝癌衍生誘導生長因子(hepatoma-derived growth factor; HDGF)，為一個新發現的生長因子，由人類肝癌細胞株 Huh-7的培養液中所純化出，HDGF的過度表現在和許多種類的癌症的初期診斷相關連，包括了黑色素癌。然而，在黑色素癌化過程中，有哪些機制會影響HDGF過度表現至今尚未明瞭。在本論文中，外加HDGF蛋白質會刺激B16-F10黑色素癌細胞侵入和形成聚落的能力。利用腺病毒當載體攜帶HDGF和干擾型HDGF (HDGF-RNAi)，在B16-F10細胞中產生作用後，會改變HDGF的表現。由實驗中發現，HDGF的過度表現會刺激B16-F10細胞增生、侵入及形成非附著性聚落的能力，而HDGF-RNAi則會抑制而產生相反的結果。在轉移到肺部的動物實驗中，利用尾靜脈注射的方式，打入有過度表現HDGF的黑色素癌細胞至老鼠體內，HDGF的過度表現能增加轉移的能力及減少HDGF的表現能有效的抑制轉移。相同的，在原位的黑色素癌動物實驗中，利用皮下注射的方式打入有過度表現HDGF的黑色素癌細胞至老鼠體內，HDGF的過度表現會促進腫瘤的生長且HDGF的減少能有效的抑制響腫瘤生長的速度及大小。組織切片染色和免疫螢光染色的分析中，在HDGF的過度表現下，黑色素癌有細胞增生、血管新生及細胞凋亡的情形。更多的證據顯示，HDGF的過度表現會促進從原位腫瘤轉移到淋巴結及肺的能力。最後，由結果中得知，HDGF的過度表現會改變nuclear factor kappa B (NFκB)， Akt phosphorylation，及其上下游之轉錄因子，例如: PI3K, PTEN, IκB及其家族 IKKα, IKKβ, IKKγ在黑色素癌中的活性，此外，在基因傳送後，HDGF的過度表現也能增加MITF及HIF1α在黑色素癌中的含量。且改變EMT對於黑色素癌的影響。綜合許多結果，HDGF的增加會促進黑色素癌的生長及轉移，利用血管新生的方式來增加癌細胞的生存。此外，中斷HDGF的表現對於黑色素癌的治療在現今是個新穎的方式。

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression during melanoma carcinogenesis remains unclear. In this study, adding exogenous HDGF stimulated the invasion and colonies formation of B16-F10 melanoma cells. Adenovirus vectors encoding HDGF and HDGF-RNAi were generated and characterized to up- and down-regulated HDGF expression in B16-F10 melanoma cells. It was found that HDGF overexpression stimulated the proliferation, invasiveness, anchorage-independent growth of B16-F10 melanoma cells whereas HDGF knockdown exerted opposite effects. In lung-metastasis model, intravenous injection of HDGF-overexpressing melanoma cells resulted in increased metastasis while HDGF-downregulated melanoma cells caused decreased metastasis. Similarly, in primary melanoma model, subcutaneous injection of HDGF-overexpressing melanoma cells enhanced while HDGF-downregulated melanoma cells reduced the tumor burden in mice. Histological analysis revealed increased tumor proliferation and neovascularization with concomitant reduction of apoptosis in HDGF-overexpressing melanoma. Moreover, HDGF-overexpressing melanoma also exhibited enhanced propensity to metastasize from the primary tumors to lymph node and lung. Finally, it was found that HDGF overexpression increased nuclear factor kappa B (NFκB) activities and Akt phosphorylation up and down stream alternation like PI3K, PTEN, IκB and it’s subunit IKKα, IKKβ, IKKγ in melanoma cells. It also found that HDGF overexpression influenced MITF and HIF1α in melanoma after gene delivery. HDGF also altered EMT changes like E,N-cadherin, vimentin, and β,γ-catenin. The present study provides conclusive evidence that HDGF upregulation promotes the growth and metastasis of melanoma by promoting the survival and vascularization. Besides, HDGF knockdown may constitute a novel strategy for melanoma control.

CONTENTS Page
Abstract in Chinese 5
Abstract in English 6
Abbreviations 8
Introduction 9
Specific Aims 15
Materials and Methods 17
Results 29
Discussion 38
Figures and Legends 43
Appendix 76
References 81

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