中文摘要
腫瘤溶解性病毒是一種嶄新的癌症治療法。病毒的治療效果主要是靠病毒本身溶解的特性，和腫瘤細胞與正常細胞間分子的差異性。為了達到選擇性的毒殺腫瘤，其中一種策略是將病毒的基因改造，使其無法在正常的細胞內複製，卻仍能夠溶解腫瘤細胞。在此研究中，我們建構了一E1B缺失性腺病毒，Ad5WS1，並探討它對具有不同p53活性的膀胱癌和肝癌細胞的毒殺性。相對於野生型的p53細胞，Ad5WS1能對突變型的p53癌細胞產生嚴重的細胞溶解。經由感染複製缺陷性腺病毒攜帶報告性基因gal的實驗顯示，這種差異性並非是細胞株對腺病毒的感受性不同所致。為了進一步證實Ad5WS1和p53的關聯性，我們送入一突變性的p53質體於野生型的p53膀胱癌細胞中，此舉能大大的增強Ad5WS1對其毒殺的能力。此外，B型肝炎病毒X蛋白質能夠藉由附著p53而抑制p53的作用。我們將一表現B型肝炎病毒X蛋白質的質體送入野生型的p53 Chang liver細胞中，X蛋白質大量表現於細胞質中，此舉能使大部分的p53滯留於細胞質中妨礙其進入細胞核執行調控的功能。p53轉錄功能的喪失，使得細胞對病毒的感受性大增。此外，在實驗動物中施打Ad5WS1至腫瘤中，能抑制膀胱癌和B型肝炎病毒X蛋白質表現肝癌細胞的生長。如分別將病毒併用阻滞血管新生的抑制劑或化學治療劑，對腫瘤細胞的生長更具有加成性的抑制作用。為了增加病毒對肝癌細胞的標的毒殺能力，我們將此E1B缺失性腺病毒E1A基因的啟動子改為一特異性在肝細胞表現的啟動子，TTRP，構築出對肝細胞具特異性的腫瘤溶解性病毒，Ad5WS2。 相較於Ad5WS1，Ad5WS2能更有效率地毒殺帶有突變型p53的人類和鼠類的肝癌細胞，然對於同樣帶有突變型p53的非肝癌細胞則否。對於具野生型的p53 Chang liver細胞亦不具有毒殺的能力。此外，在腫瘤內施打Ad5WS2能更有效的降低實驗動物中肝癌細胞的生長能力，但對肺癌細胞則無效。我們更進一步建立小鼠惡性肝癌腹水的模式證明：Ad5WS2能經由腹腔注射的方式較Ad5WS1更專一地複製於腹腔的腫瘤中，以降低腹水的形成和延緩小鼠的壽命。若合併使用化學治療劑，更能增強此抑制作用。總而言之，本論文發現E1B缺失性腺病毒對膀胱癌和B型肝炎病毒X蛋白質表現的肝癌細胞具有治療的能力，利用具肝細胞特異性的E1B缺失性腺病毒能更有效率和安全地治療肝癌和與惡性肝癌相關的腹水。

英文摘要
Oncolytic adenoviruses are an innovative class of promising cancer therapeutics. The use of oncolytic adenoviruses as a cancer therapeutic is dependent on the lytic properties of the viral cycle, and the molecular differences between tumor cells and nontumour cells. One strategy for achieving the desired tumor selectivity is to introduce loss-of function mutation in viral genes that are essential for viral replication in normal cells but not tumor cells. In this study, we constructed an E1B-55 kD-deleted adenovirus, designated Ad5WS1, and examined its cytolytic effect on human bladder cancer cell lines and hepatocellular carcinoma (HCC) cell lines with various p53 statuses. Ad5WS1 caused more severe cytolytic effect and replicated more efficiently in tumor cells carrying mutant p53 compared with cells with wild type p53. Cells susceptible to lysis caused by Ad5WS2 were not attributable to their greater infectability by adenovirus. Introduction of dominant negative p53 into p53 wild type bladder cancer cells, BFTC-905, rendered them more susceptible to Ad5WS1-induced cytolysis. Since hepatitis B virus X protein (HBx) can bind and inactivate p53, we next introduce HBx into Chang liver cells and examine their susceptibility to Ad5WS1-induced cytolysis. Expression of HBx in Chang liver cells changed the location of p53 from the nucleus to the cytoplasm, which was mostly coincided with the location of HBx in the cytoplasm. Disruption of p53 transcription activity by HBx in Chang liver cells rendered them susceptible to infection with Ad5WS1. Furthermore, in vivo animal models, Ad5WS1 exerted antitumor effect in BALB/c mice bearing HBx-expressing HCC and TCC-SUP bladder tumor xenografts, which could be augmented when combined with replication-defective adenoviral vector encoding an angiogenic inhibitor or chemotherapeutic agent cisplatin, respectively. To generate a replication-competent adenovirus with specificity for HCC, we constructed Ad5WS2, an E1B-55 kD-deleted adenovirus with its E1A gene driven by the liver-specific human transthyretin promoter (TTRP). Ad5WS2 replicated efficiently as wild type adenovirus in human and murine HCC cells carrying mutant p53, but was much attenuated in non-liver cancer cells carrying mutant p53 and in liver cells with wild-type p53. Intratumoral injection of Ad5WS2 into murine ML-1 HCC, but not into LL-2 lung cancer led to effective antitumour effect. Moreover, peritoneal administration of Ad5WS2 in BALB/c mice bearing ascites hepatoma resulted in more significant prolongation of survival than Ad5WS1, which may be attributable to reduced hepatic toxicity and more specific tumor-restricted replication of Ad5WS2. The antitumor effect of Ad5WS2 could be enhanced when combined with cisplatin in the ascites model. Taken together, the E1B-deleted adenovirus may have therapeutic potential for the treatment of bladder cancer and HCC with loss of p53 transcription activity or with HBx expression. The utility of thansthyretin promoter in the generation of E1B-deleted adenovirus may be more safe and effective for the treatment of primary HCC and malignant ascites associated with HCC.

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