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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蕭超隆 | |
| dc.contributor.author | Tzu-Hsiang Lai | en |
| dc.contributor.author | 賴子詳 | zh_TW |
| dc.date.accessioned | 2021-06-08T03:38:34Z | - |
| dc.date.copyright | 2019-07-23 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-07-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21578 | - |
| dc.description.abstract | 小分子藥物經常使用在癌症治療中，儘管這些小分子在進入人體後並不具有標靶性。具有療效的小分子通常會引起副作用。為了避免這些副作用，科學家們嘗試發展出一種可以將藥物精準地標靶至特定癌化組織的運送載體。類病毒顆粒蛋白是由病毒的衣殼蛋白(capsid)所構成，為一種奈米等級大小且具有自體組裝特性的蛋白質載體。其表面可經由化學修飾或基因工程表達標靶胜肽(targeting peptide)和抗原表位(epitope)。目前為止，Qβ、MS2和HBVc類病毒顆粒蛋白常被研究應用在疫苗領域或是做為運輸載體。在本研究中，我利用AP205類病毒顆粒蛋白包裹兩種抗生素，阿黴素(doxorubicin)和新黴素(neomycin)。將兩種抗生素和T4多聚核苷酸酶(T4 PNK)催化之硫代磷酸化髮夾核糖核酸(phosphorothioated RNA hairpin)利用胺基硫基交聯劑(amine-to-sulfhydryl crosslinker)進行共價鍵結。髮夾核糖核酸對AP205次蛋白衣(capsomere)有親和作用力，可以藉此將鍵結抗生素的髮夾核糖核酸包裹在類病毒顆粒蛋白中。我成功地在試管內組裝了包裹抗生素的AP205類病毒顆粒蛋白，並稱之為AP205 VLPs dox和AP205 VLPs neo。但是AP205 VLPs dox和AP205 VLPs neo在癌細胞實驗中沒有毒殺效果的表現。實驗數據表示造成細胞毒殺能力低落的原因可能是髮夾核糖核酸的硫代磷酸化效率過低，導致在類病毒顆粒蛋白中的藥物攜帶量不足。在未來的實驗中，我們須進一步研究如何增加阿黴素和新黴素對髮夾核糖核酸的鍵結效率。 | zh_TW |
| dc.description.abstract | Nowadays, small molecular drugs are used for chemotherapy to treat cancer diseases despite that these small molecules are non-targeting drugs. However, all efficacious drugs have adverse side effects. In order to avoid these unwanted hazards, scientists dedicate efforts to develop a carrier, which is capable of delivering drugs to target cancer cells precisely instead of anonymously. VLPs (Virus like particles) is a nanoscale self-assembled protein-based cage that is composed of only capsid from virus. The surface of capsid is tolerated with chemical modifications or genetic engineering to present the targeting peptides and epitopes. By far, the Qβ, MS2, and HBVc VLPs have been extensively studied in application of vaccine and drug delivery. In this study, the AP205 VLPs are used to encapsulate two small molecular drugs, doxorubicin (dox) and neomycin (neo), through the RNA hairpin-capsomere interactions. The encapsulated RNA hairpin is phosphorothioated by T4 PNK reaction, and then covalently coupled with dox or neo through amine-to-sulfhydryl crosslinker. The AP205 VLPs dox and AP205 VLPs neo are packed successfully in vitro. However, both the in vitro packed VLPs show low cytotoxicity towards cancer cells, which indicates that low coupling efficiency of dox/neo to the RNA hairpin, contributing to a low level of compound loading within the VLPs. The dox/neo and RNA hairpin coupling require further investigations in future works. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T03:38:34Z (GMT). No. of bitstreams: 1 ntu-108-R06B46012-1.pdf: 2837866 bytes, checksum: 073720cc28cbad9a55343edca06571db (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 論文口試委員會審定書 i
誌謝 ii 中文摘要 iii ABSTRACT iv CONTENTS v LIST OF FIGURES vii CHAPTER 1: INTRODUCTION 1 1.1 The virus like particles derived from the bacteriophage AP205 2 1.2 The pros and cons of using AP205 VLPs as a drug delivery material 5 1.3 Antibiotics 7 1.4 The aim of this study 10 CHAPTER 2: MATERIALS AND METHODS 11 2.1 Construction of expression vector 11 2.1.1 AP205 coat protein plasmid construction 11 2.1.2 RNA hairpin plasmid construction 11 2.2 Expression and Purification of AP205 VLPs 12 2.3 The AP205 VLPs coat protein (dimers) purification 14 2.3.1 Disassembly of AP205 VLPs 14 2.3.2 The AP205 VLPs coat protein (dimers) purification 14 2.4 In vitro transcription of RNA hairpin 15 2.5 Biochemical modification of the 5’-RNA hairpin 15 2.6 RNA hairpin-antibiotic coupling 16 2.7 In vitro assembly of AP205 VLPs cargo 17 2.8 The transmission electron microscopy images of AP205 VLPs 18 CHAPTER 3: RESULTS 19 3.1 Purification of AP205 VLPs coat protein dimers 19 3.2 In vitro transcription of RNA hairpin 23 3.3 Identification of RNA hairpin-antibiotic coupling 25 3.4 The assembly efficiency of AP205 VLPs in vitro 29 3.5 Characterization and purification of in vitro assembly AP205 VLPs cargo 31 3.6 Cytotoxicity of AP205 VLPs cargo towards cervical cancer cells line 34 CHAPTER 4: DISCUSSION 36 4.1 The RNA hairpin-doxorubicin coupling efficiency test 36 4.2 Non-covalent loading of doxorubicin into AP205 VLPs 38 4.3 An alternative covalent loading of doxorubicin into AP205 VLPs 39 4.4 Summary and future works 41 SUPPLEMENTARY MATERIALS 43 LITERATURE CITATIONS 44 | |
| dc.language.iso | en | |
| dc.title | 試管內組裝AP205類病毒顆粒蛋白作為抗生素藥物傳遞平臺 | zh_TW |
| dc.title | An antibiotic delivery platform using in vitro packed AP205 VLPs | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張震東,冀宏源 | |
| dc.subject.keyword | AP205類病毒顆粒蛋白,類病毒顆粒蛋白,試管內組裝,小分子藥物運送,阿黴素,新黴素, | zh_TW |
| dc.subject.keyword | AP205 virus like particles,VLPs,in vitro assembly,small molecular drug delivery,doxorubicin,neomycin, | en |
| dc.relation.page | 52 | |
| dc.identifier.doi | 10.6342/NTU201901487 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2019-07-17 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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