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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李建國(Chien-Kuo Lee) | |
| dc.contributor.author | Chan-Yu Chang | en |
| dc.contributor.author | 張展瑜 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:22:40Z | - |
| dc.date.available | 2021-11-08 | |
| dc.date.available | 2022-11-24T03:22:40Z | - |
| dc.date.copyright | 2021-11-08 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-09-15 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80936 | - |
| dc.description.abstract | 樹突細胞是連結先天性和適應性免疫系統的重要抗原呈現細胞，樹突細胞可以分化成傳統型樹突細胞(cDC)和漿狀樹突細胞(pDC)，其表面有不同的抗原並擁有不同的生理功能。然而，對於cDC和pDC的轉錄調控大部分仍未清楚。我們先前的研究指出Mef2c的表現在pDC高於cDC，且條件基因剔除Mef2c會使pDC發育受損。Flt3配體已知對於樹突細胞的發育調控是重要的。然而，Flt3訊息傳遞路徑以及磷酸化酶級聯如何調控Mef2c的活性仍不清楚。在此，我們的結果顯示在脾臟內的樹突細胞表現六號同功型Mef2c。我們用含有Mef2c結合位的klf2和α-catenin 螢光素酶報導質體來研究Flt3訊息傳遞路徑的下游是如何調控Mef2c的活性。當過度表達Flt3受體和Mef2c時，螢光速酶活性會隨著FL配體的加入而增加。此外，p38和ERK5已知會磷酸化Mef2c，在Ba/F3細胞株過度表達p38和ERK5會使螢光速酶活性上升，並隨著FL配體的刺激而有更顯著地增加。除此之外，加入p38抑制物可以降低螢光素酶的活性，顯示p38可能為Flt3訊息傳遞路徑下游中可以調節Mef2c活性的主要磷酸化酶。Mef2c的T293、T300、S387位點已知會被p38或ERK5磷酸化，在293T或Ba/F3中，磷酸模擬(S/TE/D)或磷酸失活(S/TA)突變並不會改變顯著地改變Mef2c活性。我們也找到另一個在S3962的磷酸化位點，其並不受p38所調控，不過這個位點在樹突細胞發育之中所扮演的角色仍然不清楚。Mef2c不足在永生化的造血前驅及幹細胞株中，會導致runx2和tcf4調控pDC發育重要的基因表現量下降。利用含Mef2c結合位的runx2增強子的螢光速酶活性分析實驗說明FL訊息傳遞路徑也會透過調控Mef2c活性來調控runx2的表現。統整上述結果，Flt3訊號可透過p38磷酸酶上調Mef2c活性進而調控runx2來控制pDC的發育。此外，我們也用Mef2c多肽鏈作為致免疫物並生產出高親和力的大鼠多株抗體，提供高靈敏度的工具以偵測某些細胞中低表現量的Mef2c。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-24T03:22:40Z (GMT). No. of bitstreams: 1 U0001-1309202112142300.pdf: 4857281 bytes, checksum: 0ed33c6a45ea327feec65f7085447e23 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "致謝………………………………………………………………...…......i 摘要……………………………………………………………...……….ii Abstract………………………………………………………………... iii Abbreviation……………………………………………………………..v Contents……………………………………………………………..…vii Chapter I Introduction……………………………...…………………. 1 1.1 Dendritic cells………………….……………..………………….2 1.2 Fms-like tyrosine kinase 3 (Flt3), a key cytokine receptor supporting DC development……………………………………..4 1.3 Transcriptional networks regulating DC development…………..5 1.4 Myocyte enhancer factor 2C (Mef2c) ....………......….…………7 1.5 Regulation of Mef2c activity……………………….…………….8 1.6 Rationale………………………………………………………..10 Chapter II Materials and Methods…………………….……..……….12 2.1 Cell line culture……………………………….……………...…13 2.2 DNA constructs for luciferase assay……………………………14 2.3 Antibodies, and inhibitors………………………...…………….15 2.4 Midi preparations…………………………...…………………..16 2.5 Transfection of cells…………………………………………….17 2.6 Flow cytometry………………….……………………………...18 2.7 Luciferase reporter assay…………………………………….....18 2.8 In vitro culture and differentiation of iHSPC…………………..19 2.9 RT-QPCR……………………………………………………….19 2.10 Immunoprecipitation and western blot analysis……………….19 2.11 Statistical analysis……………………………………………..20 2.12 Rabbit polyclonal antibody production………………………..21 2.13 Rat polyclonal antibody production…………………...………22 Chapter III Results……………………….……………………………23 3.1 Expression of Mef2c isoform in primary spleen cells…….…….24 3.2 Flt3 signaling pathway regulates Mef2c activity in HEK 293T cells and Ba/F3………………………………………………………..24 3.3 Flt3 signaling pathway upregulates Mef2c activity through p38 in Ba/F3 cell line……………………………………………...……25 3.4 phosphorylation of S59, T293, T300 and S387 of Mef2c activity is not involved in FL-regulated Mef2c activity…………………26 3.5 Flt3 signaling pathway induces S396 phosphorylation in Mef2c.27 3.6 Mef2c has six potential tyrosine phosphorylation sites…….…..27 3.7 Mef2c deficiency downregulates runx2 and tcf4 expression in vitro…...………………………………………………………28 3.8 Mef2c regulate Runx2 reporter activity…………………………29 3.9 Antigen preparation for Mef2c antibody production……………29 3.10 Rabbit Mef2c polyclonal antibody production………………..30 3.11 Rat Mef2c polyclonal antibody production……………………31 Chapter IV Discussion………………………………………………....33 4.1 Mef2c isoform expression in immune cells…………………….34 4.2 p38 and ERK5 regulate Mef2c activity…………………………34 4.3 Flt3 signaling pathway induces Mef2c S396 phosphorylation…36 4.4 Rabbit and rat Mef2c polyclonal antibody production………….37 Chapter V Figures………………………………….…………………..38 5.1 Expression of Mef2c isoforms in different splenocytes…………39 5.2 Flt3 signaling pathway regulates Mef2c activity in HEK 293T cells and Ba/F3 cells………………………………………………..41 5.3 p38 MAPK regulates mef2c dependent reporter activity in HEK 293T cells and Ba/F3 cells……………………………………43 5.4 p38 inhibitor reduces Mef2c dependent reporter activity in Ba/F3 cells………………………………………...…………………45 5.5 The effects of Mef2c phosphorylation on mef2c activity in HEK 293T cells and Ba/F3 cells……………………………………47 5.6 Flt3 signaling pathway induces phosphorylation of p38 and Mef2c of S396 in mouse Ba/F3 cell line……………………………..50 5.7 Flt3 signaling pathway induces Mef2c S396 phosphorylation and translocation into nuclear in human Reh cell line…………….51 5.8 potential tyrosine phosphorylation sites in Mef2c are highly conserved among different species……………………………52 5.9 Mef2c deficiency downregulates tcf4 and runx2 expression in vitro……………………………………………………...……54. 5.10 Flt3 signaling pathway upregulates a Runx2 reporter activity through p38 MAPK in 293T and Ba/F3 cell line………………55 5.11 The effects of Mef2c phosphorylation on mef2c activeity under Runx2 reporter system in Ba/F3 cell line……………………..59 5.12 Working model in controlling FL-regulated Mef2c phosphorylation for pDC development……………………….60 5.13 Immunogenicity prediction, GST-fusion construction, overexpression and purification of Mef2c-GST fusion protein for generating anti-Mef2c antibody………………………………61 5.14 Analysis of Mef2c rabbit polyclonal antibodies………………66 5.15 Production and analysis of rat polyclonal antibody against Mef2c…………………………………………………………70 Chapter VI References…………………………..….………………….73" | |
| dc.language.iso | en | |
| dc.subject | Flt3配體 | zh_TW |
| dc.subject | ERK5磷酸化酶 | zh_TW |
| dc.subject | 漿狀樹突細胞 | zh_TW |
| dc.subject | Mef2c | zh_TW |
| dc.subject | p38磷酸化酶 | zh_TW |
| dc.subject | Mef2c | en |
| dc.subject | ERK5 kinase | en |
| dc.subject | p38 kinase | en |
| dc.subject | Flt3 ligand | en |
| dc.subject | plasmacytoid dendritic cell | en |
| dc.title | Mef2c的磷酸化在FL調控的漿狀樹突細胞發育中所扮演的角色 | zh_TW |
| dc.title | The Role of Mef2c Phosphorylation in FL-Regulated DC Development | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 嚴仲陽(Hsin-Tsai Liu),徐嘉琳(Chih-Yang Tseng) | |
| dc.subject.keyword | 漿狀樹突細胞,Mef2c,Flt3配體,p38磷酸化酶,ERK5磷酸化酶, | zh_TW |
| dc.subject.keyword | plasmacytoid dendritic cell,Mef2c,Flt3 ligand,p38 kinase,ERK5 kinase, | en |
| dc.relation.page | 78 | |
| dc.identifier.doi | 10.6342/NTU202103141 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-09-16 | |
| 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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