微小RNA(miRNA)具可同時調控多個致病基因表現的特點，成為目前極具發展性的肝癌基因治療標的之一。成功的miRNA治療方法所需之基因遞送系統需要具有高專一性、高傳遞效率和長期表達治療性miRNA的特性。然而於全身性給藥的基因治療系統中，基因治療載體常被肺和血管內皮細胞非專一性的吸收，因而產生許多副作用和降低治療效率，且藥物效期極短。為改善此安全性問題，我們發展了創新的微小RNA治療方法。我們首先發現miR-200c和miR-126分別僅在正常肺細胞和靜脈上皮細胞中大量表達，而於肝細胞中低表達，此特性使我們能夠基於不同的miRNA表達特徵來設計miRNA結合位作為SB轉位系統之開關。接著，我們整合高選擇性之SB100X系統與miR-122表達系統至單一質體中以使我們的藥物實現高選擇性表現SB轉位酶和長期表達治療性miR-122的目標。雖於轉染時因低轉染效率導致無法長效表現miR-122，然經改用桿狀病毒載體運送後GFP+ 細胞比例長達1個月皆維持在20%，miR-122表現量也明顯較短效組與對照組高。未來將透過多功能奈米載體裝載之並抑制肝癌於小鼠中的發生。相信本治療策略能為肝癌治療提供一有效且高專一性又安全的平台。

miRNAs can regulate multiple genetic and epigenetic events simultaneously. Thus, miR-122 serves as potent nucleic acid therapeutics to treat liver cancer. Successful miRNA therapy requires suitable delivery systems with high specificity, high efficiency and long-term expression of therapeutic miRNAs. However, in vivo nanoparticle administration inevitably results in nucleic acid entry into non-hepatocyte cells, leading to leaky miRNA expression gene and possible toxicity. It also has a problem with short effect time. To improve safety and efficiency, we develop a novel miRNA therapy. We build the switch that exploits the Sleeping Beauty (SB) transposon-based system signature in hepatocytes. We first uncovered that miR-200c and miR-126 are exclusively expressed in normal lung cell and vein endothelial cell as well as lowly expressed in liver cancer cells, respectively, which allowed us to engineer a switch based on distinct miRNA expression signature, namely miRNA binding site technique. We have developed SB100X based system to enable hepatic specific and long-term expression of therapeutic miR-122, and then incorporated the entire device into a single plasmid. Next, we will validate the function of our plasmid in vitro. Although the low transfection efficiency leaded to low expression level, we still prove our hypothesis successfully through transduction of the baculovirus system. As a result, the GFP+ population kept around 20% at least one month, and the miR-122 expression level in long-term group was significantly higher than such in the short-term group and Mock group. In the future, we will evaluate antitumor effects after systemic administration of therapeutic SB vectors expression miR-122 loaded in the nanoparticle in the liver cancer models. We expect that this highly efficient and specific miRNA delivery system can serve as a potent anti-tumor therapeutic modality and a translational platform for clinical applications.

摘要 I
Abstract II
目錄 III
圖目錄 V
表目錄 VI
第一章 文獻回顧 1
1-1 miRNA 系統 1
1-1-1 微小RNA (microRNAs; miRNAs) 1
1-1-2 以miRNAs為基礎的治療方式 2
1-2 肝細胞癌 (Hepatocellular carcinoma, HCC) 3
1-2-1 背景 3
1-2-2 miR-122在生理功能及在病程中所扮演的角色 4
1-3 長效表現系統 Sleeping beauty (SB) 6
1-4 結合miRNA與合成生物學發展新的治療型載體 7
1-4-1 調控基因表達的合成開關 (Synthetic switch) 7
1-5 多功能奈米載體 Multifunctional nanoparticles (NP) 8
1-6 研究動機 8
第二章 實驗材料與方法 15
2-1 細胞培養 15
2-2 質體建構 15
2-3 轉染策略 17
2-4 桿狀病毒轉導策略 18
2-5 實驗分析方法 19
2-5-1 及時偵測同步定量聚合酶連鎖反應分析(qRT-PCR analysis) 19
2-5-2 流式細胞儀分析螢光表現量 20
2-5-3 西方墨點法 21
第三章 結果與討論 26
3-1 辨認適合區分肝與非肝組織之微小 RNA表現圖譜 26
3-2 以合成生物學之switch概念設計具選擇性之SB100X轉殖基因表現平台 27
3-3 miR-122 表現系統於Hep3B中功能性分析 28
3-4 轉染SB100X-m122系統於Hep3B細胞之基因嵌入效率與基因表現之延長 29
3-5 利用桿狀病毒轉導SB100X-m122系統於Hep3B細胞調控miR-122 30
3-6 不同Promoter對於桿狀病毒的轉導SB100X-m122系統於Hep3B細胞調控miR-122的影響 31
3-7 小結 31
第四章 討論與未來展望 40
4-1 針對選擇性SB100X之設計概念討論 40
4-2 關於HUVEC與NCI-H520表現量較低之疑慮 41
4-3 低轉染效率導致miR-122與GFP基因無法長效表現 42
4-4 病毒轉導與質體轉染之抉擇 43
4-5 啟動子相互干擾 43
4-6 miR-122的上調對肝之負面影響與對於其他非肝細胞之影響探討 44
4-7 CSM-S平台之延伸應用 44
第五章 參考文獻 46

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