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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 顧家綺 | |
dc.contributor.author | Chu-Nien Yu | en |
dc.contributor.author | 于主念 | zh_TW |
dc.date.accessioned | 2021-06-17T06:20:40Z | - |
dc.date.available | 2019-08-30 | |
dc.date.copyright | 2018-08-30 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-19 | |
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Lefrancois (2006). 'Novel IL-15 isoforms generated by alternative splicing are expressed in the intestinal epithelium.' Genes and Immunity 7(5): 407-416. 20. Nishimura, H., et al. (1998). 'Translational Efficiency Is Up-Regulated by Alternative Exon in Murine IL-15 mRNA.' The Journal of Immunology 160(2): 936. 21. Zhao, L., et al. (2016). 'An activation-induced IL-15 isoform is a natural antagonist for IL-15 function.' Scientific reports 6: 25822. 22. Lee, T.-L., et al. (2015). 'An Alternatively Spliced IL-15 Isoform Modulates Abrasion-Induced Keratinocyte Activation.' Journal of Investigative Dermatology 135(5): 1329-1337. 23. Chang, Y.-H. (2016). 'Effects of an alternatively spliced IL-15 isoform on IL-15-mediated signaling, T cell survival and IL-15Rα binding.' (Master’s thesis). Graduate Institute of Immunology, College of Medicine, National Taiwan University. 24. Sun, W., et al. (2016). 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M., et al. (2012). 'Mechanistic and structural insight into the functional dichotomy between IL-2 and IL-15.' Nature Immunology 13(12): 1187-1195. 31. Pettit, D. K., et al. (1997). 'Structure-Function Studies of Interleukin 15 using Site-specific Mutagenesis, Polyethylene Glycol Conjugation, and Homology Modeling.' Journal of Biological Chemistry 272(4): 2312-2318. 32. Spangler, J. B., et al. (2015). 'Insights into Cytokine–Receptor Interactions from Cytokine Engineering.' Annual Review of Immunology 33(1): 139-167. 33. Lin, Y. J., et al. (2017). 'Development of an Economical DNA Delivery System by 'Acufection' and its Application to Skin Research.' Journal of Visualized Experiments. Exp. (122), e55206. 34. Kono, T., et al. (1990). 'Murine interleukin 2 receptor beta chain: dysregulated gene expression in lymphoma line EL-4 caused by a promoter insertion.' Proceedings of the National Academy of Sciences 87(5): 1806. 35. Macauley-Patrick, S., et al. (2005). 'Heterologous protein production using the Pichia pastoris expression system.' Yeast 22(4): 249-270. 36. Moraga, I., et al. (2014). 'Multifarious Determinants of Cytokine Receptor Signaling Specificity.' Advances in Immunology. F. W. Alt, Academic Press. 121: 1-39. 37. Gröne, A. (2002). 'Keratinocytes and cytokines.' Veterinary Immunology and Immunopathology 88(1): 1-12. 38. Juráňová, J., et al. (2017). 'The role of keratinocytes in inflammation.' Journal of Applied Biomedicine 15(3): 169-179. 39. Yano, S., et al. (2003). 'Interleukin 15 induces the signals of epidermal proliferation through ERK and PI 3-kinase in a human epidermal keratinocyte cell line, HaCaT.' Biochemical and Biophysical Research Communications 301(4): 841-847. 40. Jones, A.M., et al. (2016). 'The clinical significance and impact of interleukin 15 on keratinocyte cell growth and migration.' International Journal of Molecular Medicine 38: 679-686. 41. Luo, X., et al. (2016). 'Interleukin‐15 inhibits the expression of differentiation markers induced by Ca2+ in keratinocytes.' Experimental Dermatology 25(7): 544-547. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72046 | - |
dc.description.abstract | 介白質-15 (Interleukin-15, IL-15)是四α螺旋細胞激素家族的一員,能夠調節記憶性CD8 T細胞 (Memory CD8+ T cell)、自然殺手細胞 (Natural killer cell, NK cell)、自然殺手T細胞 (Natural killer T cell, NKT cell) 以及腸道上皮內淋巴細胞 (Intestinal intraepithelial lymphocyte, IEL) 的複製和存活。介白質-15同時也是一個前發炎性細胞激素 (Proinflammatory cytokine),能夠加重人類發炎性疾病如類風濕性關節炎、乾癬、乳糜瀉等的病理狀態。本實驗室過去的研究發現,一種缺少了部分第七外顯子的IL-15選擇性剪接異構體 (以下簡稱IL-15ΔE7),具有調節皮膚免疫反應的功能,而這個效果不是因為缺少IL-15所導致。然而,這個調節作用的機制尚不清楚。為了尋找IL-15ΔE7可能的調控機制,在本論文研究中,我利用台灣國立交通大學所設計的「(PS)2蛋白質結構預測伺服器」模擬建構出IL-15ΔE7的蛋白質結構,並且用PyMOL蛋白質結構分析軟體比較IL-15和IL-15ΔE7對於IL-15受體結合的異同。結構分析結果顯示因為IL-15ΔE7缺少了對IL-15Rα的結合重要的麩胺酸 (Glutamate) 殘基,可能因此失去對IL-15Rα的結合。另外,IL-15ΔE7可能保留對於IL-2/IL-15Rβ的結合。然而,IL-15ΔE7蛋白質的構象改變 (Conformational change) 可能使其失去對於IL-2/IL-15Rγc的結合,甚至阻礙IL-15ΔE7的生物活性。得利於實驗室先前建立的酵母菌蛋白表現與純化系統,我們可以製造IL-15以及IL-15ΔE7重組蛋白,藉此在IL-2/IL-15依賴性HT-2細胞株研究IL-15ΔE7的生物活性以及其對IL-15調控反應之影響,驗證從蛋白質結構分析所獲得的結果。用飽和濃度下的IL-15ΔE7處理HT-2細胞無法引起STAT5 (IL-2/IL-15Rγc訊息傳遞途徑) 和ERK1/2 (IL-2/IL-15Rβ訊息傳遞途徑) 的磷酸化,而這跟先前的研究是一致的。另外,以IL-15ΔE7預處理接著以IL-15處理或者同時以IL-15ΔE7和IL-15共處理的實驗顯示IL-15ΔE7對IL-15媒介的訊息傳遞途徑似乎沒有拮抗效果。許多免疫學上重要的分子例如IL-2、IL-4、IL-6以及IL-15,他們的選擇性剪接異構體被認為是細胞激素訊息傳遞的天然抑制劑,會競爭原型細胞激素對於受體的結合。然而,我們尚未知道IL-15ΔE7是否為IL-15的競爭型抑制劑。為了測試這個可能性,我修改並設計了一個配體競爭性結合實驗 (Ligand competition assay)。以固定濃度的IL-15 (EC50) 和十倍序列稀釋的IL-15ΔE7共同處理HT-2細胞,再利用即時聚合酶鏈鎖反應 (Real-time PCR)檢測IL-15調控之細胞分裂和下游基因表現 (細胞週期調控基因Cdk1、抗細胞凋亡基因Bcl2) 的變化。結果顯示即使以100倍濃度的IL-15ΔE7共處理也無法抑制IL-15引起之Cdk1和Bcl2表現,這暗示IL-15ΔE7可能不會跟IL-15競爭IL-15受體結合。本論文的蛋白質結構分析預測IL-15ΔE7對於IL-15受體的結合可能會改變,因此,未來值得測量IL-15ΔE7對於各別IL-15受體次單元的結合常數 (binding constant),這有助於更精確的設計實驗以及深入了解IL-15ΔE7生物功能的分子機制。另外,IL-15ΔE7是否會透過IL-15受體引起不同的訊息傳遞途徑藉此調控IL-15的功能,尚須實驗證實。 | zh_TW |
dc.description.abstract | Interleukin-15 (IL-15) is a member of the four α-helix bundle cytokine family, which is best known to support the proliferation and survival of memory CD8+ T cells, natural killer (NK) cells, NKT cells, and intestinal intraepithelial lymphocytes (IELs). IL-15 also functions as a proinflammatory cytokine that promotes pathophysiologic state for human inflammatory diseases such as rheumatoid arthritis, psoriasis, and coeliac disease, etc. Previous study has shown that IL-15ΔE7, one variant of IL-15 generated by alternative splicing, has immune modulatory function in skin, and the effect is not caused by the lack of IL-15. However, the mechanism underlying this modulatory effect is still unknown. To find the possible mechanism underlying the IL-15ΔE7 modulation, in this thesis, I used (PS)2 (Protein Structure Prediction Server) web portal to predict the protein structure of IL-15ΔE7 and comparatively analyzed the interactions between IL-15 receptor subunits with IL-15 versus IL-15ΔE7 by PyMOL software. The results from structural analysis revealed that IL-15ΔE7 might lose its binding to IL-15Rα because it lost key glutamate residues that were responsible for receptor interaction. While the binding with IL-2/IL-15Rβ might be preserved, impaired interaction with IL-2/IL-15Rγc caused by protein conformational change could severely impede the bioactivity for IL-15ΔE7 as was well known for IL-15. Aided by the capability to generate recombinant IL-15ΔE7 protein in better quality using yeast recombinant system, the functional effects of IL-15ΔE7 on IL-15-mediated responses in IL-2/IL-15-dependent HT-2 cell line were further investigated to confirm the information derived from structural analysis. In consistent with the previous study, I demonstrated that IL-15ΔE7 treatment at saturating doses did not activate the phosphorylation of STAT5 (IL-2/IL-15Rγc signaling) and ERK1/2 (IL-2/IL-15Rβ signaling) as were induced by IL-15 in HT-2 cell line. Also, cotreatment and pretreatment experiments showed that IL-15ΔE7 had no antagonistic effect on IL-15-mediated signaling transduction. Variants of many immunologically important molecules such as IL-2, IL-4, IL-6, IL-15, generated by alternative splicing, are believed to be natural inhibitors of cytokine signaling, essentially acting as dominant negative forms of the cytokine that compete with the full-length cytokine for receptor binding. However, whether IL-15ΔE7 can also act as a competitive inhibitor of IL-15 function is unknown. To test this possibility, a “modified” ligand competition assay was performed by cotreating HT-2 cell line with fixed concentration of IL-15 (around EC50) and 10-fold serial titrated concentrations of IL-15ΔE7. IL-15-mediated responses of cell proliferation and downstream gene expression (cell cycle regulation gene Cdk1 and anti-apoptotic gene Bcl2) were measured by real-time PCR analysis. The results showed that cotreatment of IL-15ΔE7 did not block Cdk1 and Bcl2 expression even at 100 times of concentration than IL-15, which suggested that IL-15ΔE7 might not compete IL-15 for IL-15 receptor binding. Since the structural analysis have predicted the change in IL-15 receptor binding of IL-15ΔE7, it is worth to further measure the binding constant of IL-15ΔE7 to each IL-15 receptor subunits, which helps to better design the experiments and to understand the molecular mechanism of IL-15ΔE7 biological function. Also, whether IL-15ΔE7 transduces differential signaling through IL-15 receptors or that may have interfering effect on IL-15 remain to be elucidated. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T06:20:40Z (GMT). No. of bitstreams: 1 ntu-107-R05449005-1.pdf: 3057117 bytes, checksum: 76546d4af6fba4ee01e8901afacf5ad4 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii 中文摘要 iii ABSTRACT v CONTENTS vii LIST OF FIGURES x LIST OF TABLES xi Chapter 1 Introduction 1 1.1 Interleukin-15 (IL-15) 1 1.1.1 Characteristics and functions 1 1.1.2 IL-15 receptors and signal transduction 1 1.2 Control of Interleukin-15 (IL-15) expression 2 1.3 Pichia expression system 4 Chapter 2 Specific aims 6 1. To understand the differences between IL-15 and IL-15ΔE7 proteins and the precise interactions between IL-15ΔE7 and IL-15 receptors 6 2. To characterize the functions of IL-15ΔE7 and to examine whether IL-15ΔE7 affects IL-15-mediated responses in an in vitro system 7 3. To examine whether IL-15ΔE7 could compete IL-15 for IL-15 receptor binding 7 Chapter 3 Material and Methods 8 3.1 Methods 8 3.1.1 Cell line and cell culture 8 3.1.2 Expression and purification of yeast recombinant mIL-15ΔE7 8 3.1.2.1 Yeast culture and induction of yeast recombinant mIL-15ΔE7 8 3.1.2.2 Purification of yeast recombinant mIL-15ΔE7 9 3.1.3 Deglycosylation analysis of yeast recombinant mIL-15 and mIL-15ΔE7 10 3.1.4 Western blot 10 3.1.5 MTT assay 11 3.1.6 RNA extraction and quantitative real-time PCR (qRT-PCR) 11 3.1.7 Structural analysis 11 3.2 Materals 12 3.2.1 List of antibodies 12 3.2.2 List of primers 13 3.2.3 Buffers and solutions 13 Chapter 4 Results 19 4.1 Structural analysis of IL-15/IL-15ΔE7 and IL-15 receptor subunits 19 4.1.1 Structural comparison of normal IL-15 and IL-15ΔE7 protein 19 4.1.2 IL-15 receptor subunit interfaces 19 4.2 Purification and qualitative analysis of yeast recombinant mIL-15ΔE7 21 4.2.1 Purification of yeast recombinant mIL-15ΔE7 protein 21 4.2.2 Qualitative analysis of purified IL-15ΔE7 compared to IL-15 by Western blot 22 4.3 Functional analysis of purified IL-15ΔE7 22 4.3.1 IL-15ΔE7 failed to activate IL-15-mediated signaling transduction 22 4.3.2 IL-15ΔE7 had no antagonistic effect on IL-15-mediated responses 23 4.3.3 IL-15ΔE7 did not compete IL-15 for IL-15 receptor binding 24 Chapter 5 Discussion 26 5.1 Expression and purification of yeast recombinant IL-15ΔE7 26 5.2 Structural and functional analysis of IL-15ΔE7 28 FIGURES 32 TABLES 47 REFERENCE 48 | |
dc.language.iso | en | |
dc.title | 探討IL-15選擇性剪接異構體對於IL-15受體結合與IL-15調控反應之影響 | zh_TW |
dc.title | The effect of an alternatively spliced IL-15 isoform on IL-15 receptor binding and IL-15-mediated responses | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 孔祥智,李建國 | |
dc.subject.keyword | 介白質-15,選擇性剪接,蛋白質結構分析,酵母菌重組蛋白表現系統, | zh_TW |
dc.subject.keyword | Interleukin-15,alternative splicing,protein structural analysis,yeast recombinant protein expression system, | en |
dc.relation.page | 54 | |
dc.identifier.doi | 10.6342/NTU201803971 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-08-19 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 免疫學研究所 | zh_TW |
顯示於系所單位: | 免疫學研究所 |
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