肝纖維化 (Liver fibrosis) 是肝硬化的前身，在病理生理學上指出肝纖維化就是活化的成肌纖維母細胞 (myofibroblast) 及活化的肝星狀細胞 (HSCs, Hepatic Stellate Cells) 導致大量細胞外基質 (ECM) 及膠原蛋白 (collagen) 過度沈積在肝臟組織上，造成肝纖維化。近年來，抗血管新生之藥劑在治療肝纖維化上具有很高的前瞻性；此外HSCs中活化的SDF1-α (Stromal cell-derived factor α)/CXCR4 (C-X-C Chemokine receptor type 4) 軸將導致其分化 (differentiation)、增生 (proliferation) 及製造collagen I因而造成肝纖維化。
因此，我們設計一種標靶CXCR4的脂質奈米粒子，將血管內皮生長因子(VEGF, vascular endothelial growth factor) 小干擾RNA (siRNA, short-interfering RNAs) 有效地送入因餵食四氯化碳 (CCl4) 而產生肝纖維化的小鼠模型中，作為抗血管新生之藥劑；同時，我們所設計的奈米粒子將加入CXCR4的拮抗劑（antagonist）以達到標靶及抗纖維化的功效。本研究我們證明了無論在細胞實驗或是動物實驗中，標靶CXCR4的脂質奈米粒子均能有效地運送VEGF siRNA至活化的HSCs中並降低其VEGF的表現量，而因此造成纖維化肝組織中抗血管新生的效果。
此外，即使活化的HSCs會提升SDF1-α/CXCR4的表現量，奈米粒子中包覆的CXCR4拮抗劑將有效地運送至活化的HSCs中，而能抑制其分化、增生及製造collagen I的能力。綜合言之，同時結合VEGF siRNA及CXCR4 antagonist這兩種藥劑的合併療法將成為治療肝纖維化醫療上重要的里程碑。

The pathophysiology of liver fibrosis is the consequence of activation myofibroblast and hepatic stellate cells (HSCs) leading to excessive deposition of extracellular matrix (ECM) and collagen. Recently, anti-angiogenic therapy has emerged as a highly promising therapeutic strategy for treating liver fibrosis. Moreover, in HSCs, activation of SDF1-α (Stromal cell-derived factor α)/CXCR4 (Chemokine (C-X-C motif) receptor 4) axis leads to differentiation, proliferation and production of collagen I, and thus results in liver fibrosis.
Herein, we developed a CXCR4-targeted lipid-based nanoparticle (NP) formulation to specifically deliver VEGF siRNA as an anti-angiogenic agent into the fibrotic livers of CCl4-induced fibrotic models. A CXCR4 antagonist was added into NPs to serve as both a targeting moiety and an anti-fibrotic treatment. We demonstrated that CXCR4-targeted NPs could efficiently deliver VEGF siRNAs into activated HSCs in the fibrotic liver and down-regulate VEGF expression in vitro and in vivo. Efficiently delivering of VEGF siRNA to HSCs caused an anti-angiogenic effect in the fibrotic liver.
Furthermore, despite the up-regulation of SDF1-α/CXCR4 axis in activated HSCs, CXCR4 antagonist delivered by NPs effectively blocked the activation, proliferation and production of collagen I in HSCs. To sum up, co-delivering VEGF siRNA and CXCR4 antagonist as a combination treatment might be a promising approach against liver fibrosis.

總目錄
中文摘要 i
Abstract iii
總目錄 iv
圖目錄 vi
縮寫目錄 vii
第一章 緒論 1
一、研究背景 1
1.1 何謂肝硬化 1
1.2 肝硬化機制 2
1.3治療 8
1.4基因治療 10
1.4.1 Plasmid DNA(pDNA) 11
1.4.2 siRNA 12
1.4.3 以siRNA作為治療方式遇到的挑戰 13
1.4.3.1 病毒載體輸送 14
1.4.3.2 非病毒載體輸送 14
二、研究動機與目的 15
第二章 實驗材料與方法 17
2.1 所用材料 17
2.2 細胞培養 18
2.3 動物實驗 18
2.4 裝載siRNA之CXCR4-targeted NPs的製備與定性 18
2.5 免疫組織化學 20
2.6 細胞存活率分析 20
2.7 免疫螢光染色 21
2.8 西方墨點法分析 22
2.9 H&E (Hematoxylin and Eosin) 染色 23
2.10 Masson’s trichrome 染色 23
2.11 細胞攝入研究分析 24
2.12 肝攝入研究 24
2.13 統計分析方式 25
第三章 實驗結果 26
3.1 肝產生纖維化時CXCR4的表現量亦隨之增加 26
3.2 利用free AMD3100或AMD3100-NPs去抑制SDF1-α/CXCR4之結合進一步抑制HSCs的活化及增生 26
3.3 AMD-NPs將傳遞VEGF siRNAs至HSCs體外實驗及肝纖維化體內實驗，並達到顯著之基因靜默效果 27
3.4 在肝纖維化小鼠模型中，藉由AMD-NPs傳遞VEGF siRNAs進而抑制血管新生以達到抗肝纖維化之能力 29
3.5 在肝纖維化的小鼠模型中，藉由AMD-NPs傳遞VEGF siRNAs改善肝纖維化的情況 30
第四章 實驗討論與結論 32
第五章 圖表 36
第六章 參考文獻 47
 

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