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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 顧家綺 | |
| dc.contributor.author | Ping-Feng Wu | en |
| dc.contributor.author | 吳秉峰 | zh_TW |
| dc.date.accessioned | 2021-06-17T00:54:25Z | - |
| dc.date.available | 2014-03-02 | |
| dc.date.copyright | 2012-03-02 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-09-30 | |
| dc.identifier.citation | 參考文獻
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66736 | - |
| dc.description.abstract | Cytokines are important for the generation, regulation and maintenance of an effective immune response. Alternative splicing of pre-mRNA in IL-2 family cytokines has been reported and might play a role in regulating the function of the corresponding prototype cytokine. For example, interleukin-15 (IL-15) is a pleiotropic cytokine that mediates innate and adaptive immune responses. While expression of alternatively spliced IL-15 mRNAs is differentially distributed among tissues, detail mechanisms by which IL-15 activity is regulated by the splice variant remain unclear.
In this study, we generated a plasmid expressing IL-15 cDNA with a 48-nucleotide deletion in exon 7 (called IL-15ΔE7) from full length IL-15 cDNA by PCR method. IL-15 or IL-15ΔE7 protein obtained from the supernatant or lysates of COS-7 or 293T cells which were transiently transfected with a plasmid expressing IL-15 or IL-15ΔE7 gene at 24-48 hours was further analyzed by immunofluorescene, ELISA, Western blotting and IL-15 dependent cell proliferation assay. Expression of IL-15 and IL-15ΔE7 proteins running at around 18-20 kDa by gel electrophoresis was confirmed by Western blotting. Whereas IL-15 was mainly expressed in the cytoplasm, IL-15ΔE7 was restricted and expressed in the ER by immunofluorescent assays. Moreover, secretion of IL-15ΔE7 into the medium was significantly reduced compared to IL-15 by ELISA. Results from IL-15 dependent cell proliferation assays using HT-2 cells / MTT assay revealed that culture supernatant from cells transfected with full-length IL-15 cDNA contained bioactivity for HT-2 cells while the supernatant from COS-7 cells transfected with IL-15ΔE7 had no comparable IL-15 activity at all. Recently, we have also established an IL-15Rα stably expressing COS-7 cell line by retrovirus infection. The cell line will be used to study the binding of IL-15 and IL-15ΔE7 to IL-15Rα in future experiments. In this study, we have established the plasmid expressing IL-15 and IL-15ΔE7 and also have characterized the differences between IL-15 and IL-15ΔE7 by transient transfection method. Results from this and the future experiments will lead to a better understanding on how IL-15 alternative splice variant could regulate IL-15 action and its impact on IL-15Rα-mediated signaling pathway. The information will be very helpful for the development of novel strategy in treating IL-15 mediated inflammatory disorders or diseases. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T00:54:25Z (GMT). No. of bitstreams: 1 ntu-100-R98449007-1.pdf: 2836304 bytes, checksum: 2793d4c17f788456cb5c2faeb4a55369 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 口試委員審定書 i
誌謝 ii 大綱 iii Abstract iv 目錄 vi 圖目錄 ix 第一章 文獻探討 1 Part I. IL-15 1 1.IL-15的發現與蛋白質結構 1 2.IL-15的生物功能 3 3.IL-15的mRNA異構物 4 4.IL-15的轉錄與轉譯調控 4 Part II. IL-15受器與IL-15的訊息傳遞 6 1.IL-15R與IL-2R的比較 6 2.IL-15和IL-15Rα的結合 7 3.IL-15訊息傳遞 9 4.IL-15/IL-15Rα trans-presentation 9 第二章 研究目的 11 第三章 材料與方法 13 Part I. 實驗方法 13 1.細胞株與細胞培養 13 2.IL-15、IL-15ΔE7與IL-15Rα的DNA質體 13 3.PCR 多重聚合酶鏈鎖反應 13 4.以IFA 觀察IL-15及其異構物在細胞內的表現位置 14 5.以Western Blot確定細胞內的蛋白質表現 14 6.以ELISA進行IL-15的定量分析 15 7.以HT-2 assay確認IL-15的活性 15 8.建立IL-15Rα細胞株 16 Part II. 實驗材料 17 1.抗體 17 2.引子 19 3.細胞培養液 19 4.溶液 20 5.西方墨點法實驗材料與配置 22 6.其他藥劑 24 第四章 實驗結果 25 1.IL-15與IL-15ΔE7的質體建構 25 2.IL-15或IL-15ΔE7在細胞內的表現位置 26 3.比較IL-15與IL-15ΔE7的分子大小 27 4.ELISA檢測IL-15與IL-15ΔE7在轉染細胞的表現量 28 5.IL-15與IL-15ΔE7的生物活性測定 29 6.IL-15Rα細胞株的建立 31 第五章 實驗討論 33 第六章 實驗圖表 35 參考文獻 49 圖目錄 圖一：IL-15與IL-15ΔE7的質體建構。 36 圖二：IL-15或IL-15ΔE7在細胞內的表現位置。 38 圖三：IL-15與IL-15ΔE7有不同的電泳結果。 40 圖四：ELISA檢測IL-15與IL-15ΔE7在轉染細胞的表現量。 41 圖五：以HT-2/MTT檢測IL-15的生物活性。 42 圖六：GFP-RV-mIL-15Rα建構。 44 圖七：穩定表現IL-15Rα細胞株的建立。 47 | |
| dc.language.iso | zh-TW | |
| dc.subject | 細胞株 | zh_TW |
| dc.subject | 介白質-15 | zh_TW |
| dc.subject | 介白質-15異構體 | zh_TW |
| dc.subject | 介白質-15受器 | zh_TW |
| dc.subject | 反轉錄病毒 | zh_TW |
| dc.subject | IL-15R | en |
| dc.subject | cell line | en |
| dc.subject | IL-15 | en |
| dc.subject | IL-15 isoform | en |
| dc.subject | retrovirus | en |
| dc.title | 探討 IL-15 異構體對 IL-15 生物活性與 IL-15Rα 在細胞膜表現量的調控 | zh_TW |
| dc.title | Inhibition of IL-15 bioactivity and the cell surface expression of IL-15Rα by IL-15 alternatively spliced isoform | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 繆希椿,羅清維 | |
| dc.subject.keyword | 介白質-15,介白質-15異構體,介白質-15受器,反轉錄病毒,細胞株, | zh_TW |
| dc.subject.keyword | IL-15,IL-15 isoform,IL-15R,retrovirus,cell line, | en |
| dc.relation.page | 57 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-09-30 | |
| 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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