肝癌是全球最盛行的癌症之一。目前治療方式包括外科手術切除、動脈栓塞然而其治療效果不佳，因此仍需發展新的治療方針。因為肝癌具有高度血管新生的現象利用基因治療方式來持續給予血管新生抑制因子或許較易長期阻斷腫瘤血管新生進而抑制肝癌形成。Vasostatin 112 (VS112)是血管新生抑制因子vasostatin的修飾接合片段，由calreticulin的1-64和133-180所組成。在本研究中，攜帶VS 112基因重組腺病毒被生產並在同源性Sprague-Dawley大鼠N1-S1 Novikoff原位肝癌的模式中評估抑制療效。攜帶VS112基因的腺病毒顯著抑制內皮細胞的移動性及tube formation的現象，由此指出VS112基因傳送可有效抑制血管新生。然而VS 112的過量表現不影響N1-S1肝癌細胞的存活率。為了研究VS112表現對於肝癌生長的預防效果，我們以Ad-VS112 及攜帶綠螢光蛋白的腺病毒 (Ad-GFP) 去感染N1-S1細胞後植入大鼠肝臟中。14天後，在有VS112表現的大鼠相較於Ad-GFP感染的組別有顯著性的減少腫瘤發生率及腫瘤大小。再來我們研究VS112基因傳送的治療療效，在第0天直接在肝臟植入N1-S1細胞後在第1天經由靜脈給予腺病毒載體 (2 x 1010 plaques forming units) 之後在第14天觀察肝癌生長情況。 根據測量腫瘤重量和血中GOT濃度發現Ad-VS112處理過大鼠腫瘤指標相較於控制組都有明顯地減少。 組織學分析中可知Ad-VS112處理過腫瘤顯著性減少vWF-positive血管並且伴隨下降Ki-67-positive增生細胞數目和增加TUNEL-positive凋亡細胞數目。 除此之外，nuclear factor kappa B(NFκB) 和 cyclooxgenase (COXII)發炎前驅因子的表現也在Ad-VS112 處理下有效的被減緩。 總結，無論VS112 基因傳送用於預防或治療都可以有效抑制原位肝癌的形成，因而期許可以應用在未來肝癌治療上。

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. Current therapeutic approaches for HCC including surgical resection and trans-arterial embolization (TAE) remain largely ineffective, underscoring the need for development of novel therapeutic strategies. Because HCC is high vascularized, continuous administration angiogenesis inhibitor using gene therapy approach may facilitate long-term blockade tumor vasculature, thereby perturbing the growth of HCC. Vasostatin 112 (VS112) encodes an alternatively spliced fragment of angiogenesis inhibitor vasostatin, which encompasses residues 1-64 and 133-180 of calreticulin. In this study, recombinant adenovirus encoding VS112 (Ad-VS112) was generated to evaluate its potential for suppression of orthotopic Novikoff hepatoma in syngenic Sprague-Dawley (SD) rats. Adenovirus-mediated VS112 overexpression significantly inhibited the migration and tube formation of endothelial cells, indicating the anti-angiogenic potency of VS112 gene delivery. However, VS112 overexpression had no influence on the viability of N1-S1 Novikoff hepatoma cells. To investigate the prophylactic effect of VS112 expression on hepatoma growth, N1-S1 cells were infected with Ad-VS112 or adenovirus encoding green fluorescent protein (Ad-GFP) then implanted into the liver of SD rats. After 14 days, rats implanted with VS112-expressing showed significantly reduced incidence and size of hepatoma compared with those implanted with Ad-GFP-infected cells. To investigate the therapeutic efficacy of VS112 gene delivery, the SD rats were implanted with N1-S1 cells on day 0, treated with adenovirus vectors (2 x 1010 plaques forming units) via intravenous route on day 1, then sacrificed on day 14 to monitor hepatoma growth. By measuring tumor weight, it was found that Ad-VS112-treated rats exhibited significantly decreased tumor burden compared with control groups, which was in accordance with their lower serum GOT level. Histological analysis revealed a significant reduction of vWF-positive blood vessels in Ad-VS112-treated tumors, which was accompanied with a decrease in Ki-67-positive proliferating cells and an increase in TUNEL-positive apoptotic cells. Moreover, the expression of pro-inflammatory nuclear factor kappa B (NFκB) and cyclooxgenase II (COXII) was also effectively attenuated in Ad-VS112-treated hepatoma. In conclusion, prior or post VS112 gene delivery potently suppresses the growth of orthotopic hepatoma,thereby holding promises for future treatment of HCC.

CONTENTS

Abbreviations …………………………… 4
Abstract in Chinese …………………………… 5
Abstract in English …………………………… 6
Introduction …………………………… 8
Materials and Methods …………………………… 12
Results …………………………… 21
Discussion …………………………… 25
Future Perspectives …………………………… 28
References …………………………… 29
Figures and Legends …………………………… 34
Appendix ……………………………
49

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