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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 | 邱智賢 | zh_TW |
dc.contributor.advisor | Chih-Hsien Chiu | en |
dc.contributor.author | 錢彥廷 | zh_TW |
dc.contributor.author | Yen-Ting Chien | en |
dc.date.accessioned | 2024-08-07T16:58:20Z | - |
dc.date.available | 2024-08-10 | - |
dc.date.copyright | 2024-08-07 | - |
dc.date.issued | 2024 | - |
dc.date.submitted | 2024-07-27 | - |
dc.identifier.citation | Liao, Y. H. 2022. The Role of Matrix Metalloproteinase-9 (MMP-9) in LPS Triggered Liver Inflammatory Response Unpublished doctoral dissertation, National Taiwan University, Taipei.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/93747 | - |
dc.description.abstract | 肝炎是對病源入侵重要的防禦機制。一旦動物體的免疫系統持衡被打破,發炎反應將會轉變為危險的慢性肝炎,進而可能發展為肝硬化。脂多醣 (Lipopolysaccharide, LPS) 是革蘭氏陰性菌外膜的核心成分,被認為是肝炎發展的重要因素。腸道細菌過度生長和腸道通透性增加會導致過量的LPS進入肝臟,刺激促發炎細胞因子和基質金屬蛋白酶-9 (MMP9) 的表現。許多研究結果顯示,MMP9在肝臟發炎中起關鍵的保護作用。 MMP9是一種分泌性明膠酶,屬於基質金屬蛋白酶家族(MMPs)的一員。本研究旨在闡明MMP9在LPS誘導的發炎中的潛在保護作用。
我們的數據顯示,與野生型小鼠相比,MMP9基因剔除(KO)小鼠在LPS誘導後,肝臟中白介素-6(IL-6)的濃度顯著提高。推測是肝臟中的主要細胞-肝細胞和屬於巨噬細胞的Kupffer cells 中的MMP9調控了LPS誘導的發炎反應。在體外研究中,我們使用MMP9 KO RAW264.7細胞研究MMP9減緩LPS誘導發炎的機制。 在LPS誘導後,MMP9 KO細胞中的IL-6分泌量顯著高於野生型組。然而在建立了Tet-On誘導MMP9-overexpression的 RAW264.7細胞株,多西環素(doxycycline)誘導的MMP9未能緩解LPS誘導的IL-6。因此,巨噬細胞並不會透過MMP9調控IL-6的表現。接下來,透過小鼠的肝細胞株Hepa1-6細胞測試了MMP9調節肝細胞的發炎反應。我們建立了Tet-On誘導的MMP9 Hepa1-6細胞,發現多西環素誘導的MMP9降低了LPS刺激的IL-6蛋白質表現。這項發現顯示MMP9下調LPS誘導肝細胞的發炎反應。我們進一步建立了催化結構(Catalytic domain)突變的MMP9和凝血酶(hemopexin domain or PEX domain)結構缺失的MMP9,結合Tet-On系統,以闡明哪個結構在調節LPS誘導的IL-6表現中扮演關鍵角色。結果顯示,MMP9的凝血酶結構若產生缺失便無法降低IL-6的分泌。這因此,MMP9調控發炎反應,特別是IL-6,凝血酶結構域是不可或缺的。 綜上所述,我們證明了肝細胞可透過MMP9的活化來建立抗發炎反應。我們也確定了凝血酶結構對於MMP9下調發炎反應是必要的。 | zh_TW |
dc.description.abstract | Liver inflammation is one of the essential defenses against intruders. Once the homeostasis is impaired, it turns into hazardous chronic hepatitis, which may evolve into cirrhosis. Lipopolysaccharide (LPS), a central component of the outer membrane of Gram-negative bacteria, is considered a vital factor in developing hepatitis. The overgrowth of intestinal bacteria and increasing intestinal permeability cause excess LPS to enter the liver and stimulate the expressions of pro-inflammatory cytokines and matrix metalloproteinase-9 (MMP9). Numerous studies showed that MMP9 plays a critical protective role in hepatic inflammation. MMP9, a secretory gelatinase, is a matrix metalloproteinase family (MMPs) member with a zinc exonuclease. The primary function of MMP9 is the degradation and remodeling of the extracellular matrix proteins (ECMs). A recent study has suggested that MMP9 may have significant anti-inflammatory potential. The study aimed to investigate the potential protective roles of MMP9 against LPS-induced inflammation.
Our data showed that MMP9 knockout (KO) mice had significantly higher hepatic interleukin-6 (IL-6) levels than wild-type mice did after LPS induction. We hypothesized that hepatocytes and hepatic local macrophages-Kupffer cells may be the primary cells responsible for regulating LPS-induced inflammation via MMP9. In the in vitro study, we used MMP9 KO RAW264.7 cells to examine how MMP9 attenuates LPS-induced inflammatory responses. The IL-6 level in MMP9 KO cells was significantly higher than in wild-type groups after LPS induction. In addition, we established Tet-On inducible MMP9 RAW264.7 cells and found that overexpressed MMP9 through doxycycline induction did not affect mRNA and protein levels of IL-6 induced by LPS. We concluded that gene and protein expression of IL-6 in macrophages were not dependent on MMP9. Next, we tested the role of MMP9 in hepatocytes by using the mouse hepatic cell line Hepa1-6 cells. We produced Tet-On inducible MMP9 Hepa1-6 cells and found that overexpression of MMP9 attenuated the increase in IL-6 protein levels caused by LPS. This finding suggests that the MMP9 downregulates LPS-induced inflammatory responses in hepatocytes. We further established two Tet-on inducible cell lines with catalytic domain mutation and hemopexin (PEX) domain deletion to clarify which domain of MMP9 is responsible for the regulation of MMP9 in LPS-induced IL-6 expression. The results showed that the deletion of the PEX domain in MMP9 failed to attenuate IL-6 secretion. This result indicates that MMP9-regulated inflammatory response, specifically IL-6, is through the PEX domain. In summary, we demonstrated that MMP9 established anti-inflammatory function mainly in the hepatocytes. We also reported that the PEX domain of MMP9 is necessary for MMP9 to down-regulate the inflammatory response. | en |
dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2024-08-07T16:58:20Z No. of bitstreams: 0 | en |
dc.description.provenance | Made available in DSpace on 2024-08-07T16:58:20Z (GMT). No. of bitstreams: 0 | en |
dc.description.tableofcontents | 致謝 ii
中文摘要 iii Abstract iv Contents vi Table Index x Chapter 1. Introduction 1 1.1. Hepatitis 1 1.2. Lipopolysaccharide, LPS 5 1.3. Matrix metalloproteinase-9, MMP9 8 1.4. Potential roles of MMP9 in inflammation 11 1.5. Aim of the study 12 Chapter 2. Materials and Methods 13 2.1. Animals and treatments 13 2.2. ELISA 13 2.3. RNA extraction and RT-PCR analysis 14 2.4. Cell culture, infection, selection, and stimulation 15 2.5. Production and purification of lentivirus 16 2.6. CRISPR gRNA target design and cloning 17 2.7. Western blotting and zymography 19 2.8. Statistical analysis 21 3.1. MMP9 KO mice have higher levels of pro-inflammatory cytokine in the serum and liver under LPS induction 24 3.2. The dosage response of IL-6 and LPS in RAW264.7 and Hepa1-6 cells 25 3.3. MMP9 shRNA knockdown did not promote the levels of LPS-induced pro-inflammatory cytokines in RAW264.7 cells 26 3.4. MMP9 knockout affects protein levels of LPS-induced pro-inflammatory cytokines but not mRNA-level ones in RAW264.7 cells 27 3.5. Inducible MMP9 did not affect the levels of LPS-induced pro-inflammatory cytokines in RAW264.7 cells 28 3.6. Inducible MMP9 affect the protein levels of LPS-induced IL-6 in Hepa1-6 but not mRNA level 29 3.7. The PEX, not the catalytic domain, is crucial for MMP9 down-regulating LPS-induced IL-6 expressions in hepatocytes 30 Chapter 4. Discussions 51 Chapter 5. Conclusion 56 References 57 Supplementary documents 64 | - |
dc.language.iso | en | - |
dc.title | 肝細胞中第九型基質金屬蛋白酶凝血酶素結構抑制LPS誘導之IL-6分泌 | zh_TW |
dc.title | The Hemopexin domain of MMP9 attenuates LPS-induced IL-6 secretion in hepatocytes | en |
dc.type | Thesis | - |
dc.date.schoolyear | 112-2 | - |
dc.description.degree | 碩士 | - |
dc.contributor.coadvisor | 許孟傑 | zh_TW |
dc.contributor.coadvisor | Meng-Chieh Hsu | en |
dc.contributor.oralexamcommittee | 江逸凡;李月嘉 | zh_TW |
dc.contributor.oralexamcommittee | Yi-Fan Jiang;Yue-Jia Lee | en |
dc.subject.keyword | 第九型基質金屬蛋白酶,肝臟發炎反應,IL-6,凝血酶結構, | zh_TW |
dc.subject.keyword | MMP9,hepatitis,inflammation,IL-6,hemopexin domain, | en |
dc.relation.page | 64 | - |
dc.identifier.doi | 10.6342/NTU202402372 | - |
dc.rights.note | 同意授權(限校園內公開) | - |
dc.date.accepted | 2024-07-29 | - |
dc.contributor.author-college | 生物資源暨農學院 | - |
dc.contributor.author-dept | 動物科學技術學系 | - |
顯示於系所單位: | 動物科學技術學系 |
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