肝癌衍生生長因子（HDGF）在過去文獻指出會誘發許多腫瘤細胞的增殖、與加強細胞的侵入作用。而且，HDGF高度表現與患者的腫瘤進展、轉移和預後較差有關。而HDGF誘導腫瘤生長過程中，其訊息傳遞的機制尚未明確。活性氧群 (ROS)在正常生理功能下會促進細胞的生長，而在病理情況下，則會使ROS處於不穩定的狀態，其表現量過高或過低都會對細胞產生一些危害(像是老化或是癌化等等)。 HDGF促進細胞ROS的產生主要是發生在粒線體，然而HDGF誘發ROS的上升會因為粒線體電子傳遞鏈的抑制劑寡黴素存在下而被抑制。本研究目的主要是利用SK-Hep-1肝癌細胞來觀察HDGF誘導腫瘤生長過程中，ROS扮演的角色與探討其相關路徑。透過螢光偵測分析得知，ROS的表現量會隨著HDGF的劑量而有上升的趨勢（包括超氧化物陰離子和二氧化氫）。
　　HDGF誘發粒線體ROS上升與自由基清除基因表現量上升有很大的關係，包含超氧化物歧化酶2（SOD2）和過氧化氫酶 (Catalase)，但SOD1並無明顯參與其中。 HDGF會促進磷酸化Akt和核因子κB（NFB）的表現量，當外加HDGF的表面受體Nucleolin (NCL) 抗體時，會抑制HDGF誘導NFκB和SOD2的表現量，而外加NFκB的抑制劑Bay11-7082進一步證實HDGF經由NFκB路徑誘發SOD2表現。本研究證實，HDGF誘發NCL-NFκB-SOD2的訊息傳遞路徑以響應HDGF誘導的粒線體ROS升高，這可能有助於改變粒線體動力學和細胞致瘤性。

Hepatoma-derived growth factor (HDGF) has been shown to stimulate the proliferation, invasion, anchorage-independent growth in various types of cancer. In addition, HDGF overexpression is associated with tumor progression, metastasis and poor prognosis in cancer patients. The signaling and mechanism underlying HDGF-induced tumorigenesis is far from elucidation. Generation of cellular reactive oxygen species (ROS) is tightly regulated and plays a pivotal role in regulating the cellular functions as well as neoplastic transformation. The present study aimed to investigate the role of ROS production and the related pathways during HDGF-mediated tumorigenesis using SK-Hep-1 hepatoma cells. By using luminometer assay, it was observed application of HDGF dose-dependently increases the cellular ROS levels (including superoxide anion and hydrogen dioxide) in SK-Hep-1 cells. Besides, HDGF stimulated the mitochondrial ROS production, which was disrupted by electron transport inhibitor oligomycin. The HDGF-evoked mitochondrial ROS was associated with elevated expression of free radicals scavenger genes including superoxide dismutase 2 (SOD2) and catalase, but not SOD1. Exogenous HDGF protein treatment potently increased the expression of nucleolin (NCL), Akt phosphorylation and activities of nuclear factor kappa B (NFκB). Antibody neutralization of NCL abolished the HDGF-stimulated NFκB and SOD2 expression. Moreover, treatment with NFκB inhibitor reversed the HDGF-induced SOD2 upregulation. In summary, we herewith purposed that HDGF stimulates the NCL-NFκB-SOD2 signaling pathway in response to mitochondrial ROS rise, which may contribute to alterations in mitochondria dynamics and cellular tumorigenicity.

論文審定書 i
論文公開授權書 ii
誌謝 iii
摘要 iv
Abstract v
Introduction 1
Materials and methods 8
Results 14
Discussion 20
Figures and legends 23
References 36

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