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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃楓婷(Feng-Ting Huang) | |
| dc.contributor.author | Yu-Chia Pien | en |
| dc.contributor.author | 卞毓嘉 | zh_TW |
| dc.date.accessioned | 2021-06-17T03:46:19Z | - |
| dc.date.available | 2023-02-23 | |
| dc.date.copyright | 2018-02-23 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-01-29 | |
| dc.identifier.citation | Barczyk, M., Carracedo, S., & Gullberg, D. (2010). Integrins. Cell Tissue Res, 339(1), 269-280. doi:10.1007/s00441-009-0834-6
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Biomaterials, 34(35), 8909-8917. doi:10.1016/j.biomaterials.2013.07.099 Qu, M., Ren, S. C., & Sun, Y. H. (2014). Current early diagnostic biomarkers of prostate cancer. Asian J Androl, 16(4), 549-554. doi:10.4103/1008-682X.129211 Rathinam, R., & Alahari, S. K. (2010). Important role of integrins in the cancer biology. Cancer Metastasis Rev, 29(1), 223-237. doi:10.1007/s10555-010-9211-x Saini, S. (2016). PSA and beyond: alternative prostate cancer biomarkers. Cell Oncol (Dordr), 39(2), 97-106. doi:10.1007/s13402-016-0268-6 Sefah, K., Shangguan, D., Xiong, X., O'Donoghue, M. B., & Tan, W. (2010). Development of DNA aptamers using Cell-SELEX. Nat Protoc, 5(6), 1169-1185. doi:10.1038/nprot.2010.66 Shum, K. T., & Tanner, J. A. (2008). Differential inhibitory activities and stabilisation of DNA aptamers against the SARS coronavirus helicase. Chembiochem, 9(18), 3037-3045. doi:10.1002/cbic.200800491 Song, K. M., Lee, S., & Ban, C. (2012). Aptamers and their biological applications. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70151 | - |
| dc.description.abstract | 分子影像為精準醫療中一個重要的方法與工具，透過會與生物標記有專一性結合的標定探針，人們可以更準確地也更早地檢測、定位與診斷疾病。Integrin α2β1是最主要的collagen結合受體，先前的研究顯示攝護腺癌與大腸癌細胞過量表現integrin α2β1時，癌細胞更容易轉移至骨髓或肝臟這些富含collagen的環境中，因此本研究的目的是為了找出與重組integrin α2 I domain有專一結合性的適體 (aptamer)，並且將它應用在分子影像上。
SELEX (Systematic evolution of ligands by exponential enrichment) 是一個生產高親和性適體的技術，適體library會與目標分子作用以進行篩選，經過多個輪次的篩選後，存留下的適體可以與目標分子專一結合。本篇論文在SELEX中利用兩種不同的策略生產單股DNA: 非對稱PCR (asymmetric PCR) 與streptavidin beads配合鹼處理的方法。然而非對稱PCR的策略耗時又難以掌控，所以我們最終並沒有以這種方法完成篩選。我們以saturation binding assay 測試YCP-11與YCP-12這兩種候選適體的專一性結合能力，結果發現這兩種適體都不會與我們的目標蛋白質結合，更進一步，我們對不同篩選輪次的適體池 (aptamer pool) 進行定序，發現了library多樣性的流失可能是導致SELEX失敗的主因，我們認為在篩選的過程中適體的反應濃度太低或洗的條件太過嚴苛都可能造成library多樣性的流失，因此建議將篩選時的適體濃度提升至500 nM或是調整洗的條件為每次洗的體積為200 μl且一次30秒將可以改進目前的SELEX方法。 | zh_TW |
| dc.description.abstract | Molecular imaging is a powerful method and technique in precision medicine. With a labeled probe which is specific to the corresponding biomarker, people could easily detect, locate and diagnose disease in a more accurate way. Integrin α2β1 was identified as a major collagen binding receptor. Previous research indicated that prostate and colon cancers which overexpressed integrin α2β1 are more possible to metastasize to bone marrow and liver because of abundant collagen in the microenvironment. Therefore, the purpose of this research is to identify the DNA aptamer which could specifically bind to Integrin α2β1 and used as a probe in molecular imaging.
SELEX (Systematic evolution of ligands by exponential enrichment) is the technique for producing high affinity aptamer. The aptamer library would incubate with target molecules for selection. After several rounds of selection, the remaining aptamer could specifically bind to target molecule. In this thesis, two strategies for single-stranded DNA generation were used in SELEX: asymmetric PCR and streptavidin beads with alkaline treatment. However, the strategy of asymmetric PCR was time-consuming and hard to control as a result we did not finish our selection with this method. The binding affinity of two aptamer candidates, YCP-11 and YCP-12, were tested by saturation binding assay but the result showed that these aptamers were not specific binding to our target protein. Further, from the sequencing result of different round aptamer pool, we found that the losing of library diversity might be the main reason that led to the failure of SELEX. We thought that two possible reasons might cause the losing of library diversity: the concentration of aptamer was low in the selection step or the washing condition was too harsh. We suggested that increasing the concentration of aptamer to 500 nM and adjusting washing condition to 200 μl in volume with 30 seconds every time might improve the present method of SELEX. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T03:46:19Z (GMT). No. of bitstreams: 1 ntu-107-R04b22049-1.pdf: 4219017 bytes, checksum: 9f4debdaa34eac94aa38b228c5bb9ed2 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 謝辭 i
中文摘要 ii Abstract iii Chapter1 Introduction 1 1.1 Cancer and detection 1 1.2 Integrin 3 1.3 Systematic evolution of ligands by exponential enrichment (SELEX) and aptamer 7 1.4 Research purpose 10 Chapter2 Material and methods 12 2.1 Expression and purification of the recombinant human integrin α2 I domain 12 2.2 Protein analysis 13 2.2.1 Gel electrophoresis 13 2.2.2 CBR staining 14 2.2.3 Transfer 14 2.2.4 Immunoblotting 14 2.3.1 Strategy 1: in vitro selection with asymmetric PCR for single-stranded DNA generation 16 2.3.1.1 Partition of specific binding aptamer 16 2.3.1.2 PCR amplification 17 2.3.1.3 Generation of single-stranded DNA 18 2.3.1.4 Subsequent rounds of DNA aptamer selection 19 2.3.2 Strategy 2: in vitro selection with streptavidin beads and NaOH treatment for single-stranded DNA generation 19 2.3.2.1 Partition of specific binding aptamer 19 2.3.2.2 PCR amplification 20 2.3.2.3 Generation of single-stranded DNA 21 2.3.2.4 Subsequent rounds of DNA aptamer selection 22 2.3.2.5 Cloning and sequencing of enriched pools 23 2.4 6-FAM-aptamer saturation binding assay 23 2.5 in vitro experiment 24 2.5.1 Cell culture 24 2.5.2 Whole cell lysate preparation 25 Chapter3 Result 26 3.1 Expression and purification of recombinant human integrin α2 I domain 26 3.2 Development of DNA aptamers specific for integrin α2 I domain 26 3.2.1 in vitro selection with asymmetric PCR for single-stranded DNA generation 27 3.2.2 in vitro selection with streptavidin beads and NaOH treatment for single-stranded DNA generation 30 3.3 Identification of aptamer candidate for integrinα2 I domain protein 34 3.4 Measurement of the binding ability of aptamer candidates for integrin α2 I domain protein by 6-FAM aptamer saturation binding assay 35 3.5 Monitoring the evolution of aptamer pool 35 3.6 Comparison of integrin α2β1 expression level in prostate cancer and colorectal cancer cell line 36 Chapter4 Discussion 39 4.1 Possible results for losing aptamer pool diversity 39 4.2 The appearance of non-specific PCR band and possible connection with aptamer pool diversity 41 4.3 SELEX procedure 42 4.4 Current SELEX approach 44 4.5 Analyzation of integrin α2β1 expression level in different cell lines 45 Chapter5 Summary and future prospects 46 Chapter6 Figures and tables 48 Figure 1. Purification of C-terminal His-tag integrin α2 I domain from E.coli BL21 (DE3) strain 49 Figure 2. Protocol of in vitro selection with asymmetric PCR for single-stranded DNA generation 52 Figure 3. Monitoring the process of the first time SELEX selection 54 Figure 4. Monitoring the process of the second time SELEX selection 56 Figure 5. Protocol of in vitro selection with streptavidin beads and NaOH treatment for single-stranded DNA generation 59 Figure 6. Monitoring the process of the third time SELEX selection 61 Figure 7. Monitoring the process of the fourth time SELEX selection 63 Figure 8. Monitoring the process of the fifth time SELEX selection 66 Figure 9. Sequences identified and analyzed by sequence alignment program 68 Figure 10. Structure simulation of aptamer candidate 69 Figure 11. Identification of 6-FAM DNA aptamer binding ability by saturation binding assay 72 Figure 12. Expression level of integrin α2β1 in two prostate cancer cells 73 Figure 13. Expression level of integrin α2β1 in four colon cancer cells 74 Table 1. Condition of the first and the second time SELEX selection 76 Table 2. Condition of the third time SELEX selection 77 Table 3. Condition of the fourth time SELEX selection 78 Table 4. Condition of the fifth time SELEX selection 79 Appendixes 80 A. Details of primers 80 B. PCR conditions 80 C. Antibodies 82 D. Buffer 82 E. Aptamer sequence 83 F. Comparison of the condition used in several protein SELEX methods 84 G. Comparison of the condition used in several cell SELEX methods 95 H. Comparison of the condition used in several hybrid SELEX methods 100 I. Comparison of the single-stranded DNA library used in several SELEX methods 104 Oral presentation Q & A 105 Reference 110 | |
| dc.language.iso | en | |
| dc.subject | 攝護腺癌 | zh_TW |
| dc.subject | 大腸直腸癌 | zh_TW |
| dc.subject | integrin α2β1 | zh_TW |
| dc.subject | SELEX (Systematic evolution of ligands by exponential enrichment) | zh_TW |
| dc.subject | 適體 | zh_TW |
| dc.subject | 分子影像 | zh_TW |
| dc.subject | integrin α2β1 | en |
| dc.subject | prostate cancer | en |
| dc.subject | colorectal cancer | en |
| dc.subject | molecular imaging | en |
| dc.subject | aptamer | en |
| dc.subject | SELEX (Systematic evolution of ligands by exponential enrichment) | en |
| dc.title | 透過「指數富集式配體系統進化技術」篩選與Integrin α2 I domain高度專一性之適體 | zh_TW |
| dc.title | Identification of High Affinity Aptamer Specific to Integrin α2 I domain by “Systematic Evolution of Ligands by Exponential Enrichment” | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊健志,廖憶純,林晉玄 | |
| dc.subject.keyword | 攝護腺癌,大腸直腸癌,integrin α2β1,SELEX (Systematic evolution of ligands by exponential enrichment),適體,分子影像, | zh_TW |
| dc.subject.keyword | prostate cancer,colorectal cancer,integrin α2β1,SELEX (Systematic evolution of ligands by exponential enrichment),aptamer,molecular imaging, | en |
| dc.relation.page | 114 | |
| dc.identifier.doi | 10.6342/NTU201800141 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-01-30 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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