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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 蔡丰喬 | zh_TW |
dc.contributor.advisor | Feng-Chiao Tsai | en |
dc.contributor.author | 洪堂萌 | zh_TW |
dc.contributor.author | Tang-Meng Hong | en |
dc.date.accessioned | 2023-09-24T16:11:16Z | - |
dc.date.available | 2023-11-09 | - |
dc.date.copyright | 2023-09-23 | - |
dc.date.issued | 2023 | - |
dc.date.submitted | 2023-08-08 | - |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90246 | - |
dc.description.abstract | 甲狀腺眼病 (Thyroid eye disease, TED) 是一種常見於葛瑞夫茲氏症 (Graves' disease) 病患上的嚴重眼組織病變。在TED患者的眼眶中充滿脂肪及纖維化組織的現象與細胞生長激素訊號的異常活動有關。纖維母細胞生長因子 (Fibroblast growth factors, FGFs) 是一種對纖維母細胞增生和分化成脂肪細胞中扮演關鍵角色的生長因子。我們近期的研究發現,TED患者血清中FGF的水平有上升的現象,且利用FGF1刺激自眼窩中分離的纖維母細胞能夠將其轉化為前脂肪細胞 (pre-adipocytes);利用FGFR磷酸酶抑制劑則是其轉化為肌纖維母細胞 (myofibroblasts)。
由於FGFR訊息傳遞能夠藉由micro RNA let-7和miR-20a調控轉化生長因子β (TGFβR) 訊息,我們檢測了FGF是否能通過改變TGFβR訊號影響HS68纖維母細胞的分化。利用免疫墨點法,我們確認了TGF-β能夠增加細胞纖維化指標α平滑肌肌動蛋白 (α-smooth muscle actin, α-SMA) 的蛋白表現量,而同時加入FGFR磷酸酶抑制劑能夠增強這種效應,這與我們先前在眼窩纖維母細胞所觀察到的纖維化影響相符。另外在免疫螢光染色的結果也發現,抑制FGFR激酶活性極大的增強了TGF-β誘導的α-SMA應力纖維和F-actin細胞骨架的重塑,更加說明了FGFR在TGFβR訊號中扮演著關鍵角色。此外,利用慢病毒剃除FGFR1更近一步的加強了TGFβ誘導的應力纖維形成的同時卻抑制了整體細胞骨架重塑的活性。令人訝異的是,我們於免疫墨點法的結果發現到FGFR的剃除明顯地減弱了TGFβ造成的α-SMA蛋白表現的增加,與先前免疫螢光染色的發現不一致。這種細胞結構上與蛋白表現量的差異暗示著FGFR訊息對TGFβR誘導HS68纖維母細胞的分化中有著複雜的調控關係。 此研究藉由細胞模型研究TED背後的致病機制,並希望最終能夠為該疾病開發基於FGFR-TGFβR訊息調控關係的治療方法。 | zh_TW |
dc.description.abstract | Thyroid eye disease (TED) is a severe orbitopathy that frequently occurs in patients of Grave’s disease. TED patients suffer from accumulation of adipose and fibrous tissue within the orbital cavity, indicating aberrant activities of growth hormone signaling. Fibroblast growth factors (FGFs) is a growth hormone that playing a crucial role in regulating fibroblast proliferation and differentiation into adipocytes. Our recent work revealed that serum levels of FGFs were elevated in patients of TED, and that FGF1 transformed orbital fibroblasts into pre-adipocytes while FGFR inhibitors transformed them into myofibroblasts.
Since FGFR signaling regulates TGFβR signaling through microRNAs let-7 and miR-20a, we examined if FGF effects on HS68 fibroblasts were also through TGFβR modulation. Using immunoblots, we confirmed that TGF-β increased the expression of alpha smooth muscle actin (α-SMA) protein, the marker of fibrosis, and that FGFR inhibitors enhanced this effect, compatible with our previous results of FGF effects on orbital fibroblasts. Immunofluorescence also indicated that inhibition of FGFR kinase activity greatly increased TGF-β-induced α-SMA stress fiber formation and F-actin cytoskeleton remodeling, supporting the pivotal role of FGFR on TGFβR signaling. Knockdown of FGFR1 further expedited TGF-β-induced stress fiber while repressed overall actin intensity. Surprisingly, immunoblots indicated that knockdown of FGFR reduced TGF-β effects on α-SMA protein expression, deviating from our immunofluorescence findings. The discrepancy implied the complex nature of FGFR signaling on TGFβR modulation on HS68 fibroblast differentiation. This project is to elucidate the mechanisms behind TED, with the ultimate goal to develop valid FGFR-TGFβR-based therapy for the disease. | en |
dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-09-24T16:11:16Z No. of bitstreams: 0 | en |
dc.description.provenance | Made available in DSpace on 2023-09-24T16:11:16Z (GMT). No. of bitstreams: 0 | en |
dc.description.tableofcontents | Acceptance certificate i
Acknowledgement ii Abstract (Chinese) iii Abstract iv Figures and Legends ix Tables xi List of Abbreviations xii Chapter 1 Introduction 1 1.1 Overview of Thyroid eye disease 1 1.2 Growth factors signaling and TED 2 1.3 TGF-β signaling induces fibrosis in TED 3 1.4 FGFR regulated TGF-β signaling in complex manners in cell model 5 1.5 The specific aim 8 Chapter 2 Material and Methods 9 2-1 Cell culture 9 2-2 Construction of Overexpression FGFR1 KD plasmids 9 2-3 Lentivirus package 11 2-4 Cell Counting Kit-8 (CCK-8) assay 11 2-5 Immunofluorescence staining of stress fiber 12 2-6 Immunofluorescence staining of actin cytoskeleton 14 2-7 Western blot 15 2-7-1 Protein extraction 15 2-7-2 Gel-based electrophoresis (SDS-PAGE) and protein transfer 16 2-7-3 Primary and secondary antibody conjugation 16 2-7-4 Chemiluminescent immunoassay and quantification 17 Chapter 3 Results 18 3.1 FGFR signaling alters TGF-β events in HS68 fibroblast 18 3.1.1 TGF-β-induced stress fiber formation was attenuated by ligand-induced FGFR activation 18 3-1-2 Inhibition of FGFR kinase activity could further enhance TGF-β-induced stress fiber formation 22 3-2 FGFR receptor also participated in α-SMA production 24 3.2.1 Loss of FGFR1 increased stress fiber formation 24 3.2.2 Loss of FGFR1 reduced α-SMA protein expression 26 3.2.3 FGFR1 receptor was downregulated against TGF-β induction 27 3-3 The role of FGFR on TGF-β-induced actin cytoskeleton remolding 29 Chapter 4 Discussion 41 4.1 TGF-β alone induced imperceptible stress fiber in HS68 fibroblast 41 4.2 Protein level of FGFR1 and TGFβR1 receptors were regulated precisely and spatiotemporally 42 4.3 Quantification of stress fiber signal based on relative threshold of immunofluorescence signal 45 4.4 Quantification of stress fiber signal based on its cellular distribution 47 4.5 The discrepancy between immunoblotting and immunofluorescence 53 4.6 Effects of FGFR, TGFβR signaling and cell density on actin cytoskeleton remodeling on HS68 fibroblast cells 55 Chapter 5 Conclusion 56 Chapter 6 References 57 Chapter 7 Supplementary information 62 7.1 Maps of FGFR1 overexpression constructs 62 7.2 MATLAB scripts 65 | - |
dc.language.iso | en | - |
dc.title | 探討 FGFR 介導 TGFβR 訊息如何調控 Fibroblast 分化成 Adipocyte 或 Myofibroblast 的機制 | zh_TW |
dc.title | FGFR Mediates TGFβR Signaling to Switch Fibroblast Differentiation between Adipocytes and Myofibroblasts | en |
dc.type | Thesis | - |
dc.date.schoolyear | 111-2 | - |
dc.description.degree | 碩士 | - |
dc.contributor.oralexamcommittee | 涂熊林;張玉芳;鄭乃禎 | zh_TW |
dc.contributor.oralexamcommittee | Hsiung-Lin Tu;Yu-Fong Chang;Nai-Chen Cheng | en |
dc.subject.keyword | 甲狀腺眼病,纖維母細胞生長因子受體,轉化生長因子β受體,纖維母細胞分化,纖維化,應力纖維,肌動蛋白細胞骨架, | zh_TW |
dc.subject.keyword | TED,FGFR,TGFβR,fibroblast differentiation,fibrosis,stress fiber,actin cytoskeleton, | en |
dc.relation.page | 96 | - |
dc.identifier.doi | 10.6342/NTU202303790 | - |
dc.rights.note | 同意授權(全球公開) | - |
dc.date.accepted | 2023-08-09 | - |
dc.contributor.author-college | 醫學院 | - |
dc.contributor.author-dept | 藥理學研究所 | - |
顯示於系所單位: | 藥理學科所 |
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