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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80861完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 嚴仲陽(Jeffrey J. Y. Yen) | |
| dc.contributor.author | Jun-Wei Li | en |
| dc.contributor.author | 李峻緯 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:19:28Z | - |
| dc.date.available | 2022-02-15 | |
| dc.date.available | 2022-11-24T03:19:28Z | - |
| dc.date.copyright | 2022-02-15 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-02-08 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80861 | - |
| dc.description.abstract | 在慢性發炎和癌症中往往發現T細胞存在有效功能低下和抑制性受體表現量上升的狀態，稱為T細胞耗竭。由活體內系統生產耗竭性T細胞耗時且只能產生有限數量，因此活體外系統在近年逐漸被建立和驗證來克服使用活體內系統的缺點。我們成功建立一個為期五天的活體外系統用以產生似耗竭性小鼠CD8 T細胞。在此系統下藉由電穿孔送入小分子干擾核糖核酸進行基因敲落，達到調查該目標基因是否參與T細胞耗竭的目的。例如，我們發現三重敲落NR4A家族會部分回復細胞激素的產生。雖然並無在耗竭性T細胞中發現PD-1受到NR4A敲落的影響，但是在我們建立的EL4細胞模型中，由慢病毒轉染方式將小髮夾核糖核酸送入細胞來敲落NR4A1/3，結果發現佛波酯/離子黴素刺激後PD-1的表達不會上升。此外，我們也嘗試使用小分子藥物來測試是否有效預防T細胞耗竭。我們使用柏萊膜衣錠 (Dasatinib)，一種SRC激酶 (kinase) 抑制劑，發現能夠有效防止T細胞耗竭，同時NR4A家族的表達會受到抑制。進一步藉由質譜流式細胞技術 (CyTOF) 和全核糖核酸測序分析發現柏萊膜衣錠處理過的耗竭性T細胞與效應T細胞基因表徵特徵大部分一致。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-24T03:19:28Z (GMT). No. of bitstreams: 1 U0001-0602202220230300.pdf: 5003414 bytes, checksum: 9ae8e2277cb6033c7d58cf5648a42168 (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | 致謝 ii 摘要 iii Abstract iv List of figures viii Introduction 1 1 The unique transcriptional and epigenetic profile of T cell exhaustion. 1 2 NR4As are one of key factors involved in CD8 T cell exhaustion. 2 3 In vivo and in vitro models for T cell exhaustion studies 4 4 Specific aim 5 Materials and Methods 7 1 Materials 7 1.1 Antibodies 7 1.2 Buffer and reagents 8 1.3 Primers for qPCR 8 1.4 RNAi 9 1.5 Mice and cell culture 12 2 Methods 13 2.1 In vitro system generating exhausted OT-1 T cells 13 2.2 Plasmid DNA/siRNA electroporation 14 2.3 Lentiviral transduction 15 2.4 Immunoblotting 16 2.5 RNA extraction 17 2.6 RT-qPCR 17 2.7 Flow cytometry 19 2.8 RNA sequencing 20 2.9 CyTOF 21 2.10 Statistical analysis 21 Results 23 1 Establishment of EL4 T cell line as a screening system for gene manipulation in NR4A/PD-1 pathway 23 1.1 NR4A1/3 are rapidly induced in EL4 after activation. 23 1.2 Establishment of a viral transduction protocol for EL4 cell line for gene manipulation. 24 1.3 NR4A1/3 shRNA knockdowns in EL4 cells impaired PD-1 expression. …………………………………………………………………………25 2 Manipulation of NR4A family proteins in In Vitro OT-1 T cell exhaustion model 28 2.1 Establishment and characterization of the In Vitro OT-1 T cell exhaustion model 28 2.2 NR4A family proteins were also upregulated in the exhausted OT-1 cells. .......................................................................................................................30 2.3 Genetic manipulation using lentiviral transduction is inefficient in the CD8 OT-1 model 31 2.4 Genetic manipulation using siRNA electroporation in the CD8 OT-1 model 32 2.5 Decrease of NR4A family expression levels partially recovered the cytokines production 33 3 Dasatinib treatment completely reverted the T cell exhaustion phenotypes. 35 3.1 Establishment of Dasatinib treatment protocol in In Vitro T cell exhaustion model. 35 3.2 Dasatinib reversed exhausted T cells extensively resemble the effector T cells in CyTOF analysis 36 3.3 Dasatinib-treated exhausted T cells resemble effector T cells in the transcriptomic analysis 37 Discussion 40 1 Strategy switch due to technical difficulties. 40 2 The lentiviral transduction did not workout well for the primary murine T cell system. 42 3 siRNA knockdown of NR4As in OT-1 T cells did not perform a strong inhibition effect. 43 4 The major groups of in vitro induced exhausted OT-1 T cells belong to the early stage of exhaustion. 44 5 The restriction of our in vitro system 46 6 Future direction - high-throughput screening of exhaustion-associated genes and drug therapies. 47 Figures 48 Acknowledgments 78 References 79 | |
| dc.language.iso | en | |
| dc.subject | EL4細胞 | zh_TW |
| dc.subject | 柏萊膜衣錠 | zh_TW |
| dc.subject | T細胞耗竭 | zh_TW |
| dc.subject | 活體外系統 | zh_TW |
| dc.subject | 小分子干擾核糖核酸 | zh_TW |
| dc.subject | NR4A | zh_TW |
| dc.subject | 慢病毒轉染 | zh_TW |
| dc.subject | siRNA electroporation | en |
| dc.subject | dasatinib | en |
| dc.subject | lentiviral transduction | en |
| dc.subject | T cell exhaustion | en |
| dc.subject | in vitro system | en |
| dc.subject | murine CD8 T cells | en |
| dc.subject | NR4A | en |
| dc.title | 在活體外細胞培養系統中針對NR4A/PD-1訊號途徑調控T細胞耗竭 | zh_TW |
| dc.title | Targeting NR4A/PD-1 signal axis to modulate T cell exhaustion in an in vitro cell-based system | en |
| dc.date.schoolyear | 110-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 徐立中(Hsu-Yang Lin),陳世淯(Ting-Jang Lu),(Reuben Wang) | |
| dc.subject.keyword | T細胞耗竭,活體外系統,NR4A,EL4細胞,慢病毒轉染,小分子干擾核糖核酸,柏萊膜衣錠, | zh_TW |
| dc.subject.keyword | T cell exhaustion,in vitro system,murine CD8 T cells,NR4A,siRNA electroporation,lentiviral transduction,dasatinib, | en |
| dc.relation.page | 86 | |
| dc.identifier.doi | 10.6342/NTU202200305 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-02-09 | |
| 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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