MicroRNA (miRNA)透過造成基因之mRNA降解(mRNA cleavage)與阻擋蛋白質轉譯(translation)過程而影響基因表現。miR-122在肝癌細胞中，能抑制包括細胞增生、細胞週期及誘發細胞凋亡；而miR-151則透過瞄準RhoGDIA的途徑促使肝癌細胞的轉移。因此，在本研究中我們探討結合Sleeping Beauty (SB)長效系統與桿狀病毒載體分別或同時表現miR-122與miR-151 sponges，在小鼠體內實驗中抑制Mahlavu的增生和轉移。首先，我們使用四株重組桿狀病毒CdE(作為單純注射病毒的負對照組)、m122 (表現miR-122, m122組)、s151 (表現miR-151 sponge, s151組)及Dual (同時表現miR-122及miR-151 sponge, Dual組)或是磷酸鹽緩衝液(PBS組)注射至由Mahlavu細胞建立的腫瘤中，持續觀察並定期測量腫瘤大小。實驗結果發現在第30天，相對於PBS組的平均腫瘤大小，CdE組有46.9%的抑制效果；實驗組s151為70.6%，m122及Dual組的抑制效果分別為85%和86.2%。從免疫染色的實驗結果顯示，實驗組m122和Dual造成明顯的細胞凋亡並抑制細胞週期。此外，實驗組m122、s151、Dual中的腫瘤細胞轉移能力也明顯受到抑制。而肺臟轉移模型實驗的H&E染色結果顯示，實驗組m122組、s151組、Dual組則沒有看到明顯的腫瘤組織。這些結果顯示，以桿狀病毒載體在Mahlavu癌細胞內表現miR-122或/及抑制miR-151可以有效抑制腫瘤增長及轉移，也證實調控miR-122及miR-151在抑制肝癌的潛力。

MicroRNA (miRNA) regulates gene expression via mRNA degradation or preventing mRNA from being translated. MiR-122 not only reduces cell proliferation and cell cycling, but induces cell apoptosis in heptocellular carcinoma (HCC) cell lines. Moreover, miR-151 increases HCC cell migration and invasion by directly targeting RhoGDIA in HCC. Therefore, the overriding objective of this study was to develop Sleeping Beauty-based baculovirus vectors that persistently expressed a miR-151-specific sponge or harbored a cluster comprising pre-miR-122 separately or simultaneously with the system approach to inhibiting Mahlavu cell line proliferation and metastasis in vivo. First of all, we injected four types of baculovirus, named m122 (harboring a cluster comprising pre-miR-122), s151 (expressing a miR-151-specific sponge), Dual (accommodating both cassettes), CdE (harboring the d2EGFP gene under the transcriptional control of cytomegalovirus immediate-early (CMV-IE) promoter) or PBS within Sodium butyrate into the subcutaneous Mahlavu tumor. Tumor volume was then continuously monitored by measuring with calipers. The growth of tumors injected with m122, s151 or Dual was obviously inhibited. Using immunohistochemistry, we provide evidence that m122 and Dual induced HCC apoptosis and cell cycle arrest. Moreover, the ability of cell metastasis was all inhibited by m122, s151 and Dual. On the other hand, the number of lung metastases was significantly lower in group m122, s151, or Dual compared with controls. These data collectively demonstrated the feasibility of baculovirus-mediated miRNA regulation for modulating HCC cell migration and invasion, thus implicating its potential for future HCC treatment.

摘要 I
Abstract II
目錄 III
圖表目錄 V
第一章 文獻回顧 1
1.1 肝癌 (Hepatocellular carcinoma, HCC) 1
1.2 肝癌腫瘤模型 1
1.3 基因治療與MicroRNA的應用 4
1.3.1 基因治療 4
1.3.2 MicroRNA 5
1.4 桿狀病毒簡介 6
1.5 Sleeping Beauty基因轉殖系統 8
1.6 研究動機 9
第二章 實驗材料與方法 17
2.1 重組桿狀病毒的放大、濃縮與效價之量測 17
2.2 人類肝癌細胞(Mahlavu)腫瘤模型之建立 18
2.3 腫瘤生長體積之量測 18
2.4 腫瘤組織冷凍切片 19
2.5 組織切片免疫螢光染色 19
2.6 肺部腫瘤轉移模型(lung metastasis) 20
第三章 實驗結果與討論 23
3.1 陳秋伶學姊細胞實驗之數據與結果 23
3.2 人類肝癌細胞動物模型的建立 24
3.3 利用調控microRNA策略以抑制腫瘤生長之評估 25
3.4 以組織切片免疫螢光染色(IHC)評估腫瘤組織特性 26
3.4.1 細胞凋亡(apoptosis)及增生週期(cell cycle) 26
3.4.2 細胞轉移及浸潤(migration and invasion) 27
3.4.3 Epithelial-mesenchymal transition (EMT) 27
3.5 以肺臟轉移(lung metastasis)評估癌細胞在體內轉移與浸潤能力 28
3.6 結論 29
第四章 未來展望 40
4.1 增強重組桿狀病毒抑制Mahlavu肝癌細胞內的miR-151的效果 40
4.2 肺部轉移模型nodule的生成 41
第五章 參考文獻 42

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