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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李心予(Hsinyu Lee) | |
dc.contributor.author | Wei-Min Chen | en |
dc.contributor.author | 陳偉民 | zh_TW |
dc.date.accessioned | 2021-06-17T09:09:09Z | - |
dc.date.available | 2024-11-04 | |
dc.date.copyright | 2019-11-04 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-10-22 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74866 | - |
dc.description.abstract | 早衰症(Hutchinson-Gilford progeria syndrome, HGPS)為造成個體提前衰老之罕見疾病,此病症由細胞核中突變的A型合纖層蛋(Lamin A)所形成的早衰蛋白(Progerin)引起。帶有早衰蛋白的細胞,其明顯病徵為細胞核型態異變、細胞分裂緩慢、活性氧物種(Reactive oxygen species)累積及促使細胞進入細胞凋亡(Apoptosis)。水解磷酸脂(Lysophosphatidic acid, LPA)為一透過其六個受器於體內廣泛地調控生理現象之脂質生長因子,本實驗室研究中發現帶有早衰蛋白的細胞,第三型水解磷酸脂受器表現量經由大量內吞作用,並且最終由溶酶體降解而造成第三型水解磷酸脂受器所調控之細胞訊號路徑受到明顯抑制。因此,在此細胞中利用水解磷酸脂第三型受器之促進劑活化兩型受器和短髮夾 RNA(short hairpin RNA)抑制受器表現,並透過 CM-H2DCHDA 染劑和 ß-gal 染劑分別偵測活性氧物種的產生,以及細胞的老化程度。我們發現第三型水解磷酸脂受器具有能夠調控細胞中抗氧化酵素之能力,並達成延緩細胞老化的效果。藉由早衰症病患所提供之皮膚纖維母細胞反覆驗證,證實第三型水解磷酸脂受器極有可能在早衰症致病機轉中扮演重要調控角色。此外,我們於斑馬魚系統中更發現第三型水解磷酸脂受器剔除會造成早衰症。在本研究中,我們釐清第三型水解磷酸脂受器在老化過程中的的調控機轉,其中包括水解磷酸脂透過何種酵素造成活性氧化物的產生,進而影響早衰細胞中的老化。未來,專一的水解磷酸脂第三型受器之受體也期望能應用於臨床使用,增加治療老化相關疾病之可能性。 | zh_TW |
dc.description.abstract | Hutchinson-Gilford progeria syndrome (HGPS) is a rare laminopathy that produces a mutant form of prelamin A, known as Progerin, resulting in premature aging. HGPS cells show morphological abnormalities of the nuclear membrane, reduced cell proliferation rates, accumulation of reactive oxygen species (ROS), and expression of senescence markers. Lysophosphatidic acid (LPA) is a growth factor-like lipid mediator that regulates various physiological functions via activating multiple LPA G proteincoupled receptors. Here, the roles of LPA and LPA receptors in premature aging are
investigated in this study. It’s shown that the protein level of LPA3 was highly downregulated through internalization and the lysosomal degradation pathway in Progerin-transfected HEK293 cells. By treating Progerin transfected HEK293 cells with an LPA3 agonist (OMPT, 1-Oleoyl-2-O-methyl-rac-glycerophosphothionate) and performing shRNA knockdown of the Lpa3r transcript in these cells, LPA3 activation is shown to increase expression levels of antioxidant enzymes, consequently inhibiting ROS accumulation and ameliorating cell senescence. LPA3 was shown to be downregulated in HGPS patient fibroblasts through the lysosomal pathway, and it was shown to be crucial for ameliorating ROS accumulation and cell senescence in fibroblasts. Moreover, in a zebrafish model, LPA3 deficiency was sufficient to cause premature aging phenotypes in multiple organs, as well as a shorter life span. Taken together, these findings identify the decline of LPA3 as a key contributor to the premature aging phenotypes of HGPS cells and zebrafish. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T09:09:09Z (GMT). No. of bitstreams: 1 ntu-108-F03b21010-1.pdf: 9583739 bytes, checksum: 6139515ff38fa1e7b32aa728e9707e4e (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 論文口試委員審定書 I
致謝 II 中文摘要及關鍵詞 IV Abstract and Keywords V Contents VII Figure List XI Table List XIV 1. Chapter I: Lysophosphatidic Acid and Hutchinson-Gilford progeria Syndrome 1 1.1 Introduction 1 1.2 Specific Aims 5 1.3 Materials and Methods 6 1.3.1 Cell culture and pharmacological reagents 6 1.3.2 Plasmid construct, RNA interference and transfection 7 1.3.3 Western blot 7 1.3.4 Flow cytometry 8 1.3.5 Cell proliferation 9 1.3.6 Senescence-associated beta-galactosidase assay 9 1.3.7 immunofluorescent staining 10 1.3.8 Statistical analysis 10 1.4 Results 11 1.4.1 Overexpression of Progerin alters protein levels of LPA2 and LPA3 in HEK293 cells 11 1.4.2 LPA2 and LPA3 have distinct functions in regulating cell senescence in Progress HEK293 cells 12 1.4.3 LPA2 and LPA3 have distinct functions in regulating ROS in Progerin HEK293 cells 13 1.4.4 Decline of LPA3 causes ROS accumulation and cell senescence in HGPS patient fibroblasts 13 1.5 Summary 15 1.6 Figures 16 2. Chapter II: LPA3 regulates ROS level through activating Nrf2-antioxidants axis 32 2.1 Introduction 32 2.2 Specific Aims 34 2.3 Materials and methods 35 2.3.1 Measurement of mitochondrial superoxide and cellular oxygen consumption 35 2.4 Results 36 2.4.1 Decline of LPA3 causes ROS accumulation and cell senescence in HGPS patient fibroblasts 36 2.4.2 Knockdown of LPA3 sensitizes normal fibroblasts to oxidative stress 36 2.4.3 Knockdown of LPA3 sensitizes normal fibroblasts to oxidative stress 37 2.4.4 Decline of LPA3 leads to enhanced mitochondrial superoxide and reduction of mitochondrial oxidative phosphorylation 38 2.5 Summary 39 2.6 Figures 40 3. Chapter III: LPA3 is highly internalized and down-regulated through lysosomal pathway in HGPS cells 50 3.1 Introduction 50 3.2 Specific Aims 52 3.3 Materials and Methods 53 3.3.1 Determination of mRNA stability 53 3.3.2 Reverse transcription (RT) and real-time qPCR 53 3.3.3 Internalization assay and immunofluorescent staining 53 3.3.4 Luciferase assay of LPA2 promoter assay 54 3.4 Results 55 3.4.1 Neither transcription level nor mRNA stability of LPA3 is decreased 55 3.4.2 LPA3 proteins are sorted to the lysosome degradation pathway in Progerin HEK293 cells and HGPS patient fibroblasts 55 3.4.3 LPA3 proteins are highly internalized in Progerin HEK293 cells 56 3.4.4 LPA2 is upregulated in Progerin HEK293 cells through NF-κB activation 56 3.5 Summary 57 3.6 Figures 58 4. Chapter IV: Deficient of LPA3 leads to premature aging of zebrafish 66 4.1 Introduction 66 4.2 Specific Aims 68 4.3 Materials and methods 69 4.3.1 Establishment and maintenance of LPA3 knockout Zebrafish 69 4.3.2 Histological analysis of adult zebrafish 69 4.3.3 Zebrafish larvae mobility 70 4.4 Resuls 71 4.4.1 Lpa3-/- zebrafish shows premature aging phenotypes 71 4.4.2 Lpa3-/- zebrafish larvae shows higher ROS level 71 4.4.3 LPA3 deficiency caused multiple organs impairment of zebrafish 71 4.5 Summary 73 4.6 Figures 74 5. Chapter V: Conclusion and discussion of this study 78 5.1 Summary of this study 78 5.2 LPA signaling as potent factors of aging process 82 5.3 Reduction of LPA3 signaling and prevention of tumor progression 82 5.4 Alteration of LPA receptors to regulate distinct physiological functions 83 5.5 Regulation of LPA receptors expression by distinct pathways 83 5.6 Benefit and challenging for developing novel LPA3 agonists for potential therapeutics 85 5.7 Future direction: Genome instability and micronuclei in premature aging 86 6. References 88 7. Tables 98 | |
dc.language.iso | en | |
dc.title | 水解磷酸酯受器於早衰症中扮演角色之研究 | zh_TW |
dc.title | To Study the Roles of Lysophosphatidic Acid Receptors in Premature Aging | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-1 | |
dc.description.degree | 博士 | |
dc.contributor.coadvisor | 陳示國 | |
dc.contributor.oralexamcommittee | 黃偉邦,朱家瑩,蔡素宜 | |
dc.subject.keyword | 早衰症,水解磷酸酯,第三型水解磷酸酯受器,第三型水解磷酸酯受器之受體OMPT,活性氧物種,細胞老化, | zh_TW |
dc.subject.keyword | Hutchinson-Gilford progeria syndrome,Lysophosphatidic Acid,LPA3,1- Oleoyl-2-O-methyl-rac-glycerophosphothionate,Reactive oxygen species,Cell senescence, | en |
dc.relation.page | 99 | |
dc.identifier.doi | 10.6342/NTU201904228 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-10-22 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生命科學系 | zh_TW |
顯示於系所單位: | 生命科學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
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ntu-108-1.pdf 目前未授權公開取用 | 9.36 MB | Adobe PDF |
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