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利用三功能抗體武裝甲氧基聚乙二醇修飾之奈米藥物以治療富含纖維母細胞之實體腫瘤

為了解決DTX360 配件的問題，作者陳挺宇 這樣論述：

Table of ContentsTable of Contents IList of Figures IVList of Tables VList of Abbreviations VI摘要 1Abstract 21 Introduction 41.1 Current status of mPEGylated nanomedicines 41.1.1. PEGylation of nanomedicines 41.1.2. PEG dilemma 51.2 Surface modification of mPEGylated nanomedicines wit

h antibodies 61.2.1 Problems with traditional modification methods 61.2.2 Development of anti-tumor/anti-mPEG bi-specific antibodies 71.3 Tumor-associated fibroblasts–a therapeutic challenges for solid tumors 71.3.1 Roles of tumor-associated fibroblasts in tumors 71.3.2 FAP-a potential the

rapeutic target for TAFs 82 Objectives of Doctoral Dissertation 103 Materials and Methods 123.1 Hardware 123.2 Kits 123.3 Reagents 123.4 Buffers 143.5 Cells 153.6 Animals 163.7 Molecular Genetic Methods 163.7.1 DNA cloning 163.7.2 Genetic construction of engineered antibodies

163.7.3 Genetic construction of human FAP lentiviral vector 173.8 Cell Biological Methods 173.8.1 Establishment of FAP-overexpressing fibroblasts 173.8.2 Antibodies production using Expi293 expression system 183.9 Protein Biochemical Methods 183.9.1 Antibody purification 183.9.2 SDS-PAG

E analysis of TsAb and BsAbs 193.9.3 Anti-mPEG ELISA of TsAb and BsAbs 193.9.4 Flow cytometry of TsAb and BsAbs 203.10 Nanomedicine Analytical Methods 203.10.1 Optimal non-covalent modification of Lipo-Dox with TsAb or BsAbs 203.10.2 Calculation of TsAb (or BsAb) number on modified Lipo-Do

x 213.10.3 Drug release of TsAb- or BsAb-Lipo-Dox 213.10.4 Physical analysis of TsAb- or BsAb-Lipo-Dox 223.10.5 Sandwich ELISA of TsAb- or BsAb-Lipo-Dox 223.10.6 Cell-based ELISA of TsAb- or BsAb-Lipo-Dox 233.10.7 Deconvolution microscopy of drug uptake 233.10.8 In vitro cytotoxicity of

TsAb- or BsAb-Lipo-Dox 233.11 Animal Experiments 243.11.1 Pharmacokinetics of TsAb- or BsAb-Lipo-Dox 243.11.2 In vivo imaging of TsAb- or BsAb-modified Lipo-DiR 253.11.3 In vivo treatment of TsAb- or BsAb-Lipo-Dox against fibroblast-containing breast tumor 253.11.4 Toxicological evaluation

of TsAb- and BsAb-Lipo-Dox 263.12 Immunohistochemistry of TsAb or BsAb in clinical tumor tissues 263.13 Statistics 274 Results 284.1 Design and production of functional TsAb and BsAbs 284.2 Characterization of TsAb-modified Lipo-Dox 294.3 HER2- and FAP-specific cellular targeting and en

hanced cytotoxicity 304.4 Cytotoxicity against coculture of HER2+ breast cancer cells and FAP+ fibroblasts 314.5 Pharmacokinetics and in vivo tumor-targeting of TsAb-modified nanomedicines 324.6 Improved therapeutic efficacy of TsAb-Lipo-Dox against breast tumor surrounded by fibroblasts 334.

7 Specific targeting of TsAb to clinical breast cancer specimens 345 Discussion 356 Figures and Tables 417 References 628 List of Publications 70List of FiguresFigure 1. Overview of the TsAb-modified nanomedicine strategy for simultaneously eliminating tumors containing TAFs. 41Figure 2.

Genetic constructions of TsAb and BsAbs. 42Figure 3. SDS-PAGE assays of purified TsAb and BsAbs. 43Figure 4. ELISA for assessing ant-mPEG function of TsAb and BsAbs. 44Figure 5. Flow cytometric assays for assessing anti-HER2 and anti-FAP function of TsAb and BsAbs. 45Figure 6. Cellular binding a

nd free TsAb amount of TsAb-Lipo-Dox with various mPEG:TsAb modification ratios. 46Figure 7. Surface Amount of TsAb and BsAbs on the modified Lipo-Dox detected by anti-mPEG/anti-His sandwich ELISA. 47Figure 8. Cumulative release of doxorubicin from the nanomedicines. 49Figure 9. In vitro binding

ability of the nanomedicines to MCF-7/HER2, WS-1/FAP, and HaCaT cells. 50Figure 10. Fluorescence microscopy of MCF-7/HER2 cells and WS-1/FAP cells uptaking the nanomedicines. 51Figure 11. Cytotoxicity of the nanomedicines against HER2+ cells or FAP+ cells. 52Figure 12. Cytotoxicity of the nanomed

icines against coculture of MCF-7/HER2 and WS-1/FAP cells. 53Figure 13. Pharmacokinetics of BsAb- and TsAb-Lipo-Dox. 55Figure 14. Retention ratios of anti-HER2/anti-FAP/anti-mPEG TsAb and anti-HER2/anti-mPEG BsAb on the modified Lipo-Dox. 56Figure 15. In vivo tumor targeting of TsAb-Lipo-Dox in H

ER2+ breast tumor surrounded by FAP+ fibroblasts. 57Figure 16. Therapeutic efficacy of TsAb-Lipo-Dox to HER2+ breast tumor surrounded by FAP+ fibroblasts. 58Figure 17. Relative body weight of SCID mice receiving treatment with the nanomedicines. 59Figure 18. Side-effect evaluations of TsAb- or Bs

Ab-Lipo-Dox. 60Figure 19. Immunohistochemistry of human breast tumor tissue with TsAb and BsAbs. 61List of TablesTable 1. Particle size, polydispersity indices, and zeta potential of Lipo-Dox modified with TsAb or BsAbs. 48Table 2. IC50 (µg/ml) of TsAb- or BsAb-Lipo-Dox against co-culture of MCF-

7/HER2 and WS-1/FAP. 54

黑殭菌（Metarhizium anisopliae）變種間核糖體DNA內轉錄間隔區序列及二次代謝物之分析

為了解決DTX360 配件的問題，作者陳巧宜 這樣論述：

黑殭菌 (Metarhizium anisopliae (Metsch.) Sorokin ) 是一種昆蟲病原真菌，寄主範圍極廣，可感染 200 種以上的昆蟲，深具經濟及研究的價值。本研究收集國內外共計 32 株供試黑殭菌菌株，配合使用分類上常利用之核糖體核酸內轉錄間隔區 (rDNA-ITS) 定序法直接比對各菌株間核酸序列的差異度，並以鄰近接連法 (Neighbor-Joining , NJ) 及最大簡約法 (Maximum-Parsimony, MP) 建構出親緣關係樹 （phylogenetic tree）。同時本研究更進一步分析黑殭菌各菌株的二次代謝物 (secondary meta

bolites)，檢測目標以黑殭菌素 (destruxins, DTX) 和細胞鬆弛素 (cytochalasins) 為主，而此二類化合物皆各有其生物活性及醫學上之利用價值。本研究室在以往曾自南投採集所得之 Ma0407 菌株發現其可產生類細胞鬆弛素之新化合物，而購自食品工業發展研究所之菌株 BCRC35507 可產生黑殭菌素，因此由 Ma0407 及 BCRC35507 兩菌株產生之已知代謝物作為對照組，將供試菌株代謝萃取物經由高效液相層析儀 (high-performance liquid chromatography; HPLC) 分析後結果顯示 ML 13 可產生和 Ma0407 相

同的類細胞鬆弛素，而 Ma0319及 BCRC 35520 和 ARSEF 488、7488、5369 則可比對出和 BCRC 35507 相同的黑殭菌素，但各菌株間所產之黑疆菌素種類及含量均有差異，而黑疆菌素A、B、B2/E2 和 C 則普遍存在於個菌株間。經由 rDNA-ITS 定序法探討 Metarhizium 種間及變種間之親緣關係及了解變種間二次代謝物之化學多樣性 （chemodiversity），發現縱使 ITS 序列完全相同、親緣關係相近者也未必會產生完全相同之代謝物。