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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 徐立中(Li-Chung Hsu) | |
dc.contributor.author | Chun Hao Chao | en |
dc.contributor.author | 趙俊豪 | zh_TW |
dc.date.accessioned | 2021-06-16T10:47:47Z | - |
dc.date.available | 2018-09-24 | |
dc.date.copyright | 2013-09-24 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-12 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61122 | - |
dc.description.abstract | 先天免疫是宿主抵抗病原菌感染的第一道防線。TBK1為TLRs訊號傳遞下游調節干擾素產生的關鍵蛋白。在我們實驗室之前的研究中,利用酵母菌雙雜合系統篩選和TBK1有結合能力的蛋白,其中一個具結合能力的蛋白屬於分選連接蛋白家族。分選連接蛋白為細胞中囊泡運輸過程負責這種細胞膜形變的作用蛋白之一,然而,此類蛋白在TLR4的訊號傳遞上面所扮演的調控角色目前並不清楚,因此我們的研究主要想知道這個分選連接蛋白是怎麼去調節TLR4的訊號傳遞。首先,我們在293T細胞中利用免疫沉澱的實驗中確定了TBK1和此分選連接蛋白確實具有結合的能力,另外也可以在RAW 264.7細胞中利用螢光標定的方式看到兩者可以同時存在同一個區域。接著,我們在降低此分選連接蛋白的情況底下,觀察到在TLR4刺激後所調控的基因包括Ifn4a、Ifnb、Ccl5及Il6都減少了他們被轉譯的能力。另外,我們觀察到TLR4訊號傳遞最終兩個關鍵的轉路調控因子IRF3及p65入核的情況在此分選連接蛋白表現降低的情況下也受到了抑制。最後,我們檢測了幾個訊號傳遞過程中不同蛋白在此分選連接蛋白表現降低之後的活性,沒有太大的影響及變化。綜合以上的結果,我們認為此分選連接蛋白確實會調控TLR4的訊號傳遞過程,但詳細的分子機制我們還需要更多的努力來幫助我們解開這個問題。 | zh_TW |
dc.description.abstract | Innate immunity is the first line of host defense against pathogen infection. TBK1 is a key player involved in TLRs-activated type I interferon production. Previous study in our lab used yeast two-hybrid screening and obtained a TBK1-interacted protein, a sorting nexin family protein. This protein family is a machinery protein involved in vesicle trafficking. However, the function of this sorting nexin protein in the regulation of TLR4 signaling is still unknown so we here aim to study the functional role of it in the regulation of TLR4 signaling. We first confirmed the association of the sorting nexin protein and TBK1 by co-immunoprecipitation assay in 293T cells. In addition, TBK1 colocalized with it in RAW 264.7 macrophages by immunofluorescent assay. Second, the expression of Ifn4a, Ifnb, Ccl5 and Il6 mRNA was downregulationed in the sorting nexin-depleted RAW 264.7 cells upon LPS stimulation. Third, the nuclear translocation of core transcription factors in TLR4 signaling, IRF3, was decreased in the sorting nexin-depleted cells after LPS stimulation. Furthermore, LPS-induced activation of MAPKs in the sorting nexin knockdown macrophages was not change. Taken together, our data suggest that this sorting nexin regulates TLR4-mediated immune response although the detailed mechanism still awaits further investigation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T10:47:47Z (GMT). No. of bitstreams: 1 ntu-102-R00448007-1.pdf: 4628943 bytes, checksum: 14e06a2409f24bbaddbd127b1239a580 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 摘要 3
Abstract 4 Introduction 10 PAMPs, PRRs and TLRs 11 TBK1: self-activation and substrate selection 17 TLR-signaling regulation and Subcellular localization 19 Sorting nexin 1, Retromer complex and Vesicle trafficking 21 Specific aim 26 Materials and Methods 27 Antibodies and Reagents 27 Plasmids 28 Cell lines, Cell culture and Transfection 28 Lentivirus based knockdown system 29 Total cell lysate preparation 30 Immunoprecipitation 30 Subcellular fractionation 31 Immunofluorescence 32 Total RNA extraction 33 Reverse Transcription Real time quantitative PCR (RT-QPCR) 34 Statistic 34 Results 35 SNX1 associated with TBK1 and LPS induced the puncta formation of TBK1 35 Depletion of SNX1 downregulated the expression of type I interferon (IFN) and interferon responsive genes upon TLR4 activation. 36 TLR4 signaling cascade had no obvious changed whereas the nuclear translocation of IRF3 was decreased in SNX1depeleted RAW 264.7 cells after LPS stimulation. 38 TBK1 resided on late endosome/lysosome but not early endosome 39 Discussions 41 Physical interaction and colocalization between SNX1 and TBK1 41 Possible function of late endosome/lysosome-anchored TBK1 42 The expression of IL-6, a NF-κB-dependent gene, was decreased in LPS-induced SNX1 knockdown cells 45 Figures 47 Figure 1. TBK1 associated with SNX1 when overexpressedin 293T cells. 47 Figure 2. LPS induced TBK1 and SNX1 colocalization in RAW 264.7 macrophages. 49 Figure 3. The RNA expression of SNX2 was not altered in SNX1-deficienct RAW 264.7 cells 50 Figure 4. The mRNA expression of type I interferon (IRN), IFN-responsive genes, and IL-6 was decreased in SNX1 knockdown RAW 264.7 cells in response to LPS. 52 Figure 5. TLR4 signaling cascade was not obviously affected in SNX1 knockdown cells after LPS stimulation. 53 Figure 6. Nuclear translocation of IRF3 was decreased in SNX1 knockdown cells after LPS stimulation. 54 Figure 7. TBK1 was localized to late endosome/lysosome but not early endosome in RAW 264.7 cells. 55 Figure 8. TBK1 did not colocalized with SNX1 in LAMP1+ compartments in RAW 264.7 cells after LPS stimulation. 56 Figure 9. LPS increased TBK1 translocation into heavy membrane in RAW 264.7 cells. 57 Figure 10. The proposed model for the regulation of TLR4-induced immune response through SNX1 58 Supplementary figures 59 Supplementary Figure 1. TLR4 signaling pathway 59 Supplementary Figure 2. Schematic diagram of SNX1 and the mechanism of SNX1-mediated vesicle sorting. 61 Supplementary Figure 3. SNX1 was a candidate TBK1-interacting protein obtained from the yeast-two hybrid screening. 62 Supplementary Figure 4. The specificity of TBK1 antibodies used in immunoflourescent assay 63 Table1. Plasmid list 64 Table2. Primer list 65 Reference 67 | |
dc.language.iso | en | |
dc.title | 一個分選連接蛋白於TLR4調控的免疫反應上扮演的角色 | zh_TW |
dc.title | The functional role of a sorting nexin family protein in
TLR4-mediated immune response | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 繆希椿,李秀香 | |
dc.subject.keyword | 先天免疫,分選連接蛋白, | zh_TW |
dc.subject.keyword | innate immunity,sorting nexin, | en |
dc.relation.page | 76 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-08-12 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
顯示於系所單位: | 分子醫學研究所 |
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