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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃楓婷 | |
dc.contributor.author | Chern-Shiuan Liu | en |
dc.contributor.author | 劉宸亘 | zh_TW |
dc.date.accessioned | 2021-06-08T01:38:31Z | - |
dc.date.copyright | 2016-08-31 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-29 | |
dc.identifier.citation | Ashikari, D., & Takahashi, S. (2016). [The latest research of the molecular biomarker in prostate cancer]. Nihon Rinsho, 74(1), 50-54.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18887 | - |
dc.description.abstract | 根據世界衛生組織在2012年的統計報告中顯示,全球男性罹患攝護腺癌的比率佔所有癌症的第二名,且因癌症而死亡的病患人數佔所有癌症的第五名,其中侷限性攝護腺癌 (localized prostate cancer) 的病患在確診五年後的存活率高達百分之八十,而大多數造成死亡的案例是因為攝護腺癌轉移至周遭的骨骼、淋巴腺或是其他器官,所以亟需開發針對轉移型攝護腺癌的診斷平台。Integrin α2β1為type I collagen 主要的受體,且在許多癌症中會過量表現並參與癌症的惡化及轉移的路徑,所以Integrin α2β1為合適的癌症診斷的生物標記,我們的實驗目標為透過噬菌體呈現技術篩選對於integrin α2 I domain具高度專一性的胜肽,並未來將之運用於臨床檢測及分子影像。首先,我們利用C7C phage display library進行biopanning且篩選對於integrin α2 I domain有結合性的環狀胜肽,並且透過phage ELISA binding assay找到兩個與integrin α2 I domain 有結合力的目標胜肽,再利用type I collagen competition assay鑑別目標胜肽與integrin α2 I domain之間的專一性,發現只有C7 胜肽會影響type I collagen與integrin α2 I domain之間的鍵結能力。隨後,利用FITC saturation binding assay測試目標胜肽與integrin α2 I domain的dissociation constant (Kd),發現兩個目標胜肽的Kd落在μM鍵結等級區間,接著將兩個目標胜肽進行in vitro實驗。我們將具有螢光標定的C7或C8胜肽與攝護腺癌細胞PC-3或22Rv1進行反應,透過螢光訊號偵測兩個胜肽結合到攝護腺癌細胞的能力。由實驗結果發現,兩個目標蛋白對於攝護腺癌細胞鍵結強度的差異與其integrin α2β1的表現量成正相關。綜合目前的實驗結果,我們認為C7或C8胜肽在in vitro中具有與integrin α2β1鍵結的能力,而in vivo的實驗正在進行中。 | zh_TW |
dc.description.abstract | According to WHO cancer statistics in 2012, prostate cancer is the second most common cancer and the fifth leading cause of cancer death in men around the world. Eighty percent of men with the localized prostate cancer are still alive after diagnosis in 5 years, however, at the distant stage of prostate cancer, cancer that have spread to other organs, the survival rate drop dramatically and is the most causes of prostate cancer death. Therefore, it is an urgent need to develop precise and non-invasion diagnostic method for metastatic prostate cancer. The integrin α2β1 is the major type I collagen-binding receptor, and it is overexpressed in many cancer types. The importance of integrin α2β1 involved in prostate cancer metastasis has made it an appropriate biomarker for prostate cancer detection. Hence, in this thesis, the goal was to identify integrin α2β1-binding peptides and ultimately develop as the imaging agent for metastatic prostate cancer diagnosis. First, the recombinant integrin α2 I domain protein was purified and used for selection of high affinity binding peptides with the loop-constrained heptapeptides (C7C) phage library by biopanning. After measurement of the relative binding affinity by phage ELISA binding assay, two integrin α2 I domain-binding phages (C7, C8) were identified. The result of type I collagen competition assay indicated that the C7 phage, but not the C8 phage, could compete with type I collagen to bind to integrin α2 I domain. Moreover, the dissociation constant (Kd) of C7 or C8 peptide to integrin α2 I domain was determined by FITC-peptide saturation binding assay, and the Kd of C7 and C8 peptides were 7 μM and 12 μM, respectively. Furthermore, we used two peptides to target the two prostate cancer cell lines (PC-3 and 22Rv1) that have different integrin α2β1 expression level. By using FITC-labeled C7 peptide or FITC-labeled C8 peptide incubating with these two cell lines and followed by IN Cell Analyzer 2000, we demonstrated that two peptides had greater tendency of binding to PC-3 cells than 22Rv1 cells. In conclusion, C7 or C8 peptide may have potential to be developed as molecular imaging tracer in prostate cancer diagnosis. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T01:38:31Z (GMT). No. of bitstreams: 1 ntu-105-R03b22032-1.pdf: 1150141 bytes, checksum: f9ba59133ef87c0ea4770a2d31e473fa (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 謝辭(i)
中文摘要(ii) Abstract(iii) Table of Contents(v) Chapter 1 Introduction(1) 1.1 Prostate carcinoma detection(1) 1.2 Positron emission tomography(3) 1.3 Integrin α2β1 in cancer biology(4) 1.4 Phage display technology(8) 1.5 Research purpose(9) Chapter 2 Material methods(12) 2.1 Expression and purification of recombinant human integrin α2 I-s domain and integrin α2 I-l domain(12) 2.1.1 Construction of TA- integrin α2 I-s domain plasmid and integrin α2 I-l domain plasmid(12) 2.1.2 Construction of pET28b-integrin α2 I-s domain plasmid and pET28b-integrin α2 I-l domain plasmid (C-terminal His-tag) (12) 2.1.3 Transformation of pET28b-integrin α2 I-s domain plasmid or pET28b-integrin α2 I-l domain plasmid to BL21 (DE3) competent cells(13) 2.1.4 Expression and purification of integrin α2 I-s domain and integrin α2 I-l domain(14) 2.1.5 Desalting and concentration of integrin 2 I-s domain and integrin α2 I-l domain(15) 2.2 Protein analysis(16) 2.2.1 Gel electrophoresis(16) 2.2.2 CBR staining(16) 2.3 Calf type I Collagen binding assay(17) 2.4 Biopanning(18) 2.5 Phage titer analysis(20) 2.6 Extraction of phage single-stranded DNA(20) 2.7 Phage amplification(21) 2.8 ELISA (enzyme-linked immunosorbent assay) of phage binding assay(22) 2.9 Calf type I Collagen competition assay(23) 2.10 FITC-peptide saturation binding assay(24) 2.11 In vitro experiment(25) 2.11.1 Cell culture(25) 2.11.2 Integrin α2β1 expression analysis(25) 2.11.2.1 Whole cell lysate preparation(25) 2.11.2.2 Western blotting(26) 2.11.3 Peptide binding in vitro(27) 2.11.4 Fluorescence-activated cell sorting analysis(27) Chapter 3 Results(28) 3.1 Expression and purification of recombinant human integrin α2 I-s domain and integrin α2 I-l domain(28) 3.2 Comparison of ligand binding affinity by the through calf type I collagen binding assay(28) 3.3 The titer of output elution phage(29) 3.4 Identification binding ability of phage clones by phage ELISA binding assay(30) 3.5 The binding site of phage candidate on integrin 2 I-l domain(30) 3.6 Estimation of the dissociation constant of FITC peptide to integrin α2 I-l domain(31) 3.7 Determination of peptide binding specifity to prostate cancer cells(32) 3.7.1 Expression level of integrin α2β1 in two prostate cancer cell lines(32) 3.7.2 In vitro binding assay of immunofluorescence(32) 3.7.3 In vitro binding assay of fluorescence-activated cell sorting analysis(33) Chapter 4 Discussion(35) 4.1 Biopanning procedure(36) 4.2 Comparison of the binding affinity between the phage clone and the corresponding peptide(36) 4.3 Targeting integrin α2β1 in vitro(36) Chapter 5 Summary and future prospects(38) Chapter 6 Reference list(39) Figure and tables(43) Figure 1. CBR staining of purified C-terminal His-tag integrin α2 I-s and integrin α2 I-l domain(44) Figure 2. The type I collagen binding assay of integrin α2 I domain(45) Figure 3. The eluted phage titer of each round of biopanning by using integrin α2 I-l domain against with the C7C phage library(46) Figure 4. Identification of the integrin α2 I-l domain-binding-phage candidates by phage ELISA binding assay(47) Figure 5. The competition assay of integrin α2 I-l domain between calf type I collagen and candidate phage clones(49) Figure 6. The dissociation constant was determined by FITC-labeled peptides saturation binding assay and the Lineweaver-Burk double reciprocal plot(50) Figure 7. The protein level of integrin α2β1 in PC-3 and 22Rv1 prostate cancer cell lines by Western blot(53) Figure 8.Targeting of FITC-labeled C7 peptide to PC-3 or 22Rv1 prostate carcinoma cell lines(54) Figure 9. Targeting of FITC-labeled C8 peptide to PC-3 or 22Rv1 prostate carcinoma cell lines(56) Figure 10. Fluorescence-activated cell sorting analysis of FITC-labeled C7 or FITC-labeled C8 peptide binding to PC-3 cell and 22Rv1 cell lines(58) Appendix(63) A. Details of primers(64) B. PCR conditions(64) C. Antibodies(65) D. Buffer(66) E. Peptide sequences(67) F. Integrin structure illustration(67) | |
dc.language.iso | en | |
dc.title | 透過噬菌體呈現技術篩選與Integrin α2 I domain 高度專一性之胜肽 | zh_TW |
dc.title | Identification of High Affinity Peptides Specific to Integrin α2 I domain by Phage Display Technology | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 楊健志,廖憶純,陳彥榮,林貝容 | |
dc.subject.keyword | 攝護腺癌,癌症轉移,癌症生物標記,integrin α2β1,噬菌體呈現技術,分子影像, | zh_TW |
dc.subject.keyword | prostate cancer diagnosis,cancer metastasis,cancer biomarker,integrin α2β1,phage display,molecular imaging, | en |
dc.relation.page | 67 | |
dc.identifier.doi | 10.6342/NTU201603565 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-08-30 | |
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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