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研究生:黃冠瑋
研究生(外文):Huang, Kuan-Wei
論文名稱:建立半乳糖衍生物基因載體標靶ASGPR用於肝癌治療
論文名稱(外文):Establish Galactoside Derivatives Gene Vector Targeting ASGPR for Therapeutic Approach of Hepatocellular Carcinoma
指導教授:陳韻晶
指導教授(外文):Chen, Yunching
口試委員:邱健泰林淑宜
口試委員(外文):Qiu, JiantaiLin, Shu-Yi
口試日期:2018-07-12
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物醫學工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:英文
論文頁數:55
中文關鍵詞:肝癌半乳糖去唾液酸糖蛋白受體脂質磷酸鈣奈米載體β苯基半乳糖苷血管內皮生長因子之小分子干擾核糖核酸
外文關鍵詞:GalactosideLipid/calcium/phosphate nanoparticlesAsialoglycoprotein receptor (ASGPR)Hepatocellular carcinoma (HCC)Gene therapyVascular endothelial growth factor (VEGF) siRNA
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肝癌(Hepatocellular carcinoma, HCC) 細胞大量表現可辨識半乳糖基團的去唾液酸糖蛋白受體(ASGPR),可藉由半乳糖基團針對肝癌細胞作為有力的標靶物質。然而正常肝細胞亦會大量表現ASGPR,使得半乳糖同時會被兩者辨識,因此在肝癌治療中利用半乳糖基團針對肝癌作為選擇性標靶物質,仍然是需要克服的困境之一。為了藉由半乳糖基團對於肝癌建立更有力和有選擇性標靶的基因或藥物遞送,我們修飾半乳糖的結構並驗證半乳糖衍生物對肝癌細胞能產生較高的親和性。在本研究中,我們在脂質磷酸鈣(Lipid/Calcium/Phosphate, LCP) 奈米載體 (NPs) 上各別結合不同種的半乳糖衍生物,並證實經由β苯基半乳糖苷修飾的奈米粒子對於肝癌細胞和正常肝細胞表現出可區別的結合親合力。我們以L4修飾的LCP NPs (L4-LCP NPs)遞送血管內皮生長因子之小分子干擾核糖核酸 (VEGF siRNA),能夠在體外實驗及動物實驗中降低血管內皮生長因子的表現量,並在小鼠原位肝癌模型中展現有效力的抗血管生成作用,且導致顯著的腫瘤消退。實驗結果指出苯基半乳糖苷是能有希望成為標靶肝癌的半乳糖衍生物,並作為非常有潛力的基因遞送方式達到治療肝癌的目的。
Successful siRNA therapy requires suitable delivery systems with targeting moieties such as small molecules, peptides, antibodies, or aptamers. Galactose (Gal) residues recognized by the asialoglycoprotein receptor (ASGPR) can serve as potent targeting moieties for hepatocellular carcinoma (HCC) cells. However, efficient targeting to HCC via galactose moieties rather than normal liver tissues in HCC patients remains a challenge. To achieve more efficient siRNA delivery in HCC, we synthesized various galactoside derivatives and investigated the siRNA delivery capability of nanoparticles modified with those galactoside derivatives. In this study, we assembled lipid/calcium/phosphate nanoparticles (LCP NPs) conjugated with eight types of galactoside derivatives and demonstrated that phenyl β-D-galactoside-decorated LCP NPs (L4-LCP NPs) exhibited a superior siRNA delivery into HCC cells compared to normal hepatocytes. VEGF siRNAs delivered by L4-LCP NPs downregulated VEGF expression in HCC in vitro and in vivo, and led to a potent anti-angiogenic effect in the tumor microenvironment of a murine orthotopic HCC model. The efficient delivery of VEGF siRNA by L4-LCP NPs that resulted in significant tumor regression indicates that phenyl galactoside could be a promising HCC-targeting ligand for therapeutic siRNA delivery to treat liver cancer.
摘要 I
Abstract II
Acknowledgement III
Abbreviation IV
Table of Contents 1
Table of Figures 3
Table of Tables 5
Motivation and Aims 6
Introduction 7
2.1 Liver Cancer 7
2.1.1 The background of liver cancer 7
2.1.2 Treatment approaches for hepatocellular carcinoma 9
2.2 RNA Interference 10
2.3 Drug Delivery for siRNA 12
2.4 ASGPR 14
Materials and Methods 16
3.1 Materials and Equipment 16
3.1.1 Materials 16
3.1.2 Equipment 17
3.2 Cell Culture 17
3.3 Mice and Orthotopic Tumor Model Establishment 18
3.4 CuAAC conjugation to lipid-PEG 18
3.5 Preparation of Nanoparticles 24
3.6 Characterization of Nanoparticles 25
3.7 Cellular Uptake 25
3.8 Competition Assay 26
3.9 qRT-PCR 26
3.10 Western Blot Analysis 27
3.11 Tumor Growth Inhibition Study 27
3.12 Immunofluorescence 28
3.13 Hematoxylin and Eosin Staining 28
3.14 Statistics 28
Results 29
4.1 Preparation of ASGPR Targeting Nanoparticles 29
4.2 Synthesis of Alkynyl Galactosides 30
4.3 Optimization Cellular Uptake of FAM-siRNA Delivered by Galactoside-Decorated LCP NPs 33
4.4 Characteristic and Selective HCC-Targeted Ability of L4-LCP NPs 36
4.5 Angiogenesis Downregulation of VEGF siRNA-loaded L4-LCP In Vitro 42
4.6 Anti-angiogenesis Effect and Tumor Suppression of VEGF siRNA-loaded L4-LCP In Vivo 44
4.6 Tumor Suppression of VEGF siRNA-loaded L4-LCP In Vivo 46
Conclusion 49
Discussion and Future Work 51
Reference 53
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