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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 顧家綺 | |
| dc.contributor.author | Yi-Hsuan Chang | en |
| dc.contributor.author | 張毅軒 | zh_TW |
| dc.date.accessioned | 2021-05-19T17:56:14Z | - |
| dc.date.available | 2021-08-26 | |
| dc.date.available | 2021-05-19T17:56:14Z | - |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-19 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7858 | - |
| dc.description.abstract | 介白質-15 (Interleukin-15, IL-15) 是一個多效型的細胞激素，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) 及自然殺手T細胞 (Natural killer T cell, NKT cell) 的複製和存活，同時也能夠促進其他細胞激素的表現。本實驗室過去的研究中發現，一種缺少了部分的第七外顯子的IL-15選擇性剪接異構體，（以下簡稱IL-15ΔE7），無法促進IL-2和 IL-15依賴性細胞HT-2細胞的STAT5的磷酸化以及抗細胞凋亡基因bcl-2的轉錄。在本論文研究，我成功建立酵母菌蛋白表現與純化系統製造重組IL-15以及IL-15ΔE7二種蛋白，並且經過西方墨點法及快速液態層析儀 (Fast Protein Liquid Chromatography, FPLC) 確認該蛋白的活性與生化特性。為了更進一步研究IL-15ΔE7對於細胞存活的影響，利用了流式細胞儀分析分別經過IL-15以及IL-15ΔE7處理後的HT-2 細胞，藉以偵測細胞膜上磷脂絲胺酸 (phosphotidylserine, PS) 外翻及DNA斷裂的程度以檢驗細胞凋亡的情況。在以IL-15培養的情況下，大部分HT-2細胞存活並具有增殖性；然而，有很高比例的IL-15ΔE7處理過後的HT-2細胞在第12小時進行細胞凋亡，而全部的細胞在第24小時死亡，這樣的情況與去除細胞激素培養的控制組細胞類似。以IL-15ΔE7預處理接著以IL-15處理或者同時以IL-15ΔE7和IL-15處理的HT-2細胞，這兩種處理方式都不會抑制IL-15促進細胞存活與增殖的作用；此外，高濃度的IL-15ΔE7刺激也不會引起IL-15培養的細胞死亡。這些實驗闡述了雖然IL-15ΔE7無法維持HT-2細胞的存活，但是外部添加IL-15ΔE7也無法抑制IL-15調節的細胞增生。利用Micro-Western進行訊號體 (Signalosome) 分析的實驗指出，IL-15ΔE7能夠相較於IL-15引起更多的P70 S6 kinase、SrcY416、SrcY527、Syk以及GSK3的磷酸化，同時我們也觀察到更多IκB的蛋白質累積；更進一步地，我們使用傳統的西方墨點法以檢驗MAPK和SrcY416的訊息傳導並確認了IL-15ΔE7無法造成ERK的磷酸化，卻能夠造成更多的JNK、P38和SrcY416的磷酸化，我們也發現IL-15引起的IKK磷酸化在IL-15ΔE7刺激的細胞中卻是被抑制的，然而，P65的磷酸化程度在IL-15ΔE7和IL-15刺激的細胞中卻是相當的，也因此IL-15ΔE7在NF-κB訊息傳遞當中所扮演的角色需要更深入的研究。從IL-15或者IL-15ΔE7結合至IL-15Rα的情況分析及結合後對於細胞表面IL-15Rα表現的影響結果當中指出，相對IL-15而言，IL-15ΔE7以非常低的親和力結合上IL-15Rα；同時，IL-15所引起的細胞表面IL-15Rα的表現量下降，在IL-15ΔE7處理的細胞則不顯著。已知藉由非淋巴性白血球轉位呈現 (trans-presentation) IL-15至T細胞和NK細胞是對於這兩類細胞能夠發展出活化後功能的重要機制；因此，未來的實驗應該加強探討IL-15ΔE7如何藉由調控轉位呈現得以調節IL-15的功能。 | zh_TW |
| dc.description.abstract | IL-15 mediates the proliferation and survival of memory CD8+ T cells, natural killer (NK) and NKT cells as well as enhancing cytokine production. We have previously shown that an alternatively spliced IL-15 isoform that has partial deletion in exon 7 of the IL-15 gene (IL-15ΔE7) failed to activate phosphorylation of STAT5 and blocked transcription of anti-apoptotic gene bcl-2 in an IL-2/IL-15 dependent T cell line (HT-2 cells). In this thesis, I have successfully generated recombinant IL-15ΔE7 and IL-15 proteins from yeast expression system and confirmed by Fast Protein Liquid Chromatography (FPLC) and Western Blot analyses. To further investigate the effects of IL-15ΔE7 on cell survival, IL-15ΔE7-treated or IL-15-treated HT-2 cells were examined for cell apoptosis by detecting the expression of phosphatidylserine (PS) on the plasma membrane and DNA fragmentation by flow cytometric analysis. HT-2 cells were proliferative and survived in IL-15 culture. However, high percentage of cells underwent apoptosis at 12 h and all cells died at 24 h in IL-15ΔE7-treated HT-2 cells. The patterns were similar to cytokine-deprived control cells. Pretreatment of HT-2 cells with IL-15ΔE7 followed by IL-15 treatment or co-treatment of the cells with a mixture of IL-15 and IL-15ΔE7 did not blocked IL-15-mediated cell survival and proliferation. High concentration of IL-15ΔE7 treatment did not promote cell death in IL-15 treated cells. These experiments demonstrated that while IL-15ΔE7 failed to support HT-2 cell survival, exogenous addition of IL-15ΔE7 did not exert inhibitory effects on IL-15-mediated cell proliferation. Signalosome analysis by Micro-Western assay showed that IL-15ΔE7 treatment resulted in increased levels of phosphorylation of p70 S6 kinase, SrcY416, SrcY527, Syk and GSK3 compared with those in IL-15 treated cells. Increased protein level of IκB was also observed. Further analysis of the protein phosphorylation in MAPK pathways and SrcY416 by conventional Western blot confirmed that IL-15ΔE7 failed to phosphorylate ERK but enhanced phosphorylation of JNK, P38 and SrcY416. IL-15 induced phosphorylation of IKK was suppressed in IL-15ΔE7 treated cells. Since the phosphorylation levels of p65 were comparable in IL-15-treated and IL-15ΔE7-treated cells, the role of IL-15ΔE7 in NF-κB signaling pathways requires further investigation. Analysis of the binding of IL-15 or IL-15ΔE7 to IL-15Rα and the effects on the surface expression of IL-15Rα after ligand binding demonstrated that IL-15ΔE7 bound to IL-15Rα with very low affinity compared with IL-15. IL-15 triggered downregulation of surface expression of IL-15Rα was less significant in IL-15ΔE7-treated cells. Since trans-presentation of IL-15 by IL-15Rα on myeloid cells is known as an important mechanism for developing effector functions of T cells and NK cells, the role for IL-15ΔE7 in regulating IL-15 function via trans-presentation is worth of further investigation. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-19T17:56:14Z (GMT). No. of bitstreams: 1 ntu-105-R03449004-1.pdf: 3249503 bytes, checksum: 6a4e27298c102bb13d40b485e814dbab (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 誌謝 i
Abstract ii 摘要 iv Table of Contents vi List of Figures ix Chapter I. Introduction 1 Part I. Interleukin-15 (IL-15) 1 1. Characteristics 1 2. Functions 1 3. Trans-presentation 3 Part II. The interaction of IL-15 with IL-15Rα, IL-2/IL-15Rβ and γc 4 Part III. Signal transduction through IL-2/IL-15Rβ and γc 6 Part IV. NF-κB signaling pathway 6 Part V. Hypothesis of IL-15ΔE7 structure 7 Part VI. Pichia expression system 8 Chapter II. Aims of the Study 9 1. Establishment of Yeast protein expression system to generate recombinant IL-15ΔE7 and IL-15 protein 9 2. Functions of IL-15ΔE7 on cell proliferation and survival 10 3. Signalosome analysis of IL-15ΔE7 induced signaling activation in HT-2 cells 10 4. Effects of IL-15ΔE7 on the cell surface expression of IL-15Rα 11 Chapter III. Materials and Methods 12 Part I. Methods 12 1. Cell lines and cell culture 12 2. Construction of the X-33 strain expressing mouse IL-15 and IL-15ΔE7 12 2.1 Construction of pPICZαA-mIL-15(FLAG) and 12 pPICZαA-mIL-15ΔE7(FLAG) 12 2.2 Transformation of pPICZαA-mIL-15ΔE7(FLAG) and pPICZαA-mIL-15 (FLAG) into the X-33 strain 15 3. Expression and purification of yeast recombinant IL-15 or IL-15ΔE7 15 3.1 Induction of yeast recombinant IL-15 or IL-15ΔE7 15 3.2 Purification of yeast recombinant IL-15 or IL-15ΔE7 16 4. Deglycosylation analysis of yeast recombinant IL-15 and IL-15ΔE7 16 5. Coomassie blue staining of yeast recombinant IL-15 and IL-15ΔE7 17 6. Western blotting 17 7. IL-15 and IL-15ΔE7 biological assay using HT-2 cells 18 8. Flow cytometry analysis 19 8.1 Apoptosis assay 19 8.2 Immunodetection of expression of surface IL-15Rα and surface attached 19 IL-15 isoforms on IL-15Rα-COS-7 cells 19 Part II. Materials 20 1. List of antibodies 20 2. List of primers 22 3. Solutions 22 Chapter IV. Results 28 1. Expression and purification of Yeast recombinant IL-15ΔE7 and IL-15 28 1.1 Strategy of constructing yeast plasmid expressing IL-15ΔE7 or IL-15 28 1.2 Construction of recombinant IL-15ΔE7 and IL-15 expressing X33 strain 29 1.3 Expression and characterization of yeast recombinant IL-15ΔE7 and IL-15 30 1.4 FPLC analysis of yeast recombinant IL-15ΔE7 and IL-15 31 1.5 Function analysis of yeast recombinant IL-15ΔE7 and IL-15 32 2. Apoptosis and proliferation assay of IL-15ΔE7 functions 33 2.1 Apoptosis assays of IL-15ΔE7-treated HT-2 cells 33 2.2 IL-15ΔE7 failed to support HT-2 cell survival. 34 2.3 IL-15ΔE7 failed to inhibit IL-15 mediated cell survival. 35 2.4 IL-15ΔE7 fails to inhibit IL-15 mediated cell proliferation. 36 3. Signalosome analysis of IL-15ΔE7 induced signaling activation in HT-2 cells 37 3.1 IL-15ΔE7 activated alternative signaling pathways different from IL-15 mediated responses. 38 3.2 IL-15ΔE7 induced JNK, P38 and SrcY416 phosphorylation but not ERK phosphorylation. 39 3.3 IL-15ΔE7 failed to induce phosphorylation of IKK but enhances phosphorylation of P65. 39 4. Effects of IL-15ΔE7 on the cell surface expression of IL-15Rα 40 4.1 The reasons of investigating the role of IL-15ΔE7 to trans-presentation 40 4.2 IL-15ΔE7 bond to IL-15Rα with an extremely low affinity. 41 4.3 Time course analysis of surface IL-15Rα downregulation induced by IL-15ΔE7 42 4.4 IL-15ΔE7 induced surface IL-15Rα downregulation in the overnight culture. 44 Chapter V. Discussion 45 1. Establishment of Pichia expression system to generate recombinant IL-15 and IL-15ΔE7 46 2. The regulatory functions of IL-15ΔE7 on HT-2 cells 48 3. Analysis of IL-15ΔE7 mediated signaling pathways 49 4. The differences in affinity and regulation of surface IL-15Rα between IL-15 and IL-15ΔE7 51 5. The connection between in vitro experiments and P191 mouse phenotypes 53 References 57 Figures 64 Appendix 89 | |
| dc.language.iso | en | |
| dc.title | 探討介白質-15異構蛋白對於介白質-15調節訊息傳遞、T細胞存活及與介白質-15受器α結合的影響 | zh_TW |
| dc.title | Effects of an alternatively spliced IL-15 isoform on IL-15-mediated signaling, T cell survival and IL-15Rα binding | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 徐立中,李建國,孔祥智 | |
| dc.subject.keyword | 介白質-15異構蛋白,T細胞,介白質-15受器α,訊息傳遞,畢赤酵母,蛋白質純化,細胞凋亡, | zh_TW |
| dc.subject.keyword | alternatively spliced IL-15 isoform,T cell,IL-15Rα,signaling,Pichia pastoris,protein purification,apoptosis, | en |
| dc.relation.page | 90 | |
| dc.identifier.doi | 10.6342/NTU201603429 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2016-08-19 | |
| 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 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-105-1.pdf | 3.17 MB | Adobe PDF | 檢視/開啟 |
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