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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳政彰 | zh_TW |
| dc.contributor.advisor | Cheng-Chang Chen | en |
| dc.contributor.author | 劉乙珊 | zh_TW |
| dc.contributor.author | Yi-Shan Liu | en |
| dc.date.accessioned | 2026-03-13T16:43:15Z | - |
| dc.date.available | 2026-03-14 | - |
| dc.date.copyright | 2026-03-13 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-09-18 | - |
| dc.identifier.citation | [1] Si-Tahar M, Touqui L, Chignard M. Innate immunity and inflammation--two facets of the same anti-infectious reaction. Clin Exp Immunol 2009;156(2):194-8.
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J Immunol 2015;195(10):4922-32. [75] Gu ZQ, Wang HT, Li Y, Krogsaeter E, Lin AC, Lin J, Liu YS, Lin WS, Burton W, Liu ML and others. TRPML2 channel modulation by PI(3,5)P(2) and small-molecule agonists controls endosomal vesicle dynamics. Biomed Pharmacother 2025;189:118350. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/102136 | - |
| dc.description.abstract | 巨噬細胞，特別是M1表型，對於維持體內恆定以及在病原感染期間提供宿主防禦扮演關鍵角色。鈣訊號在巨噬細胞極化過程中具有核心調控功能，尤其在誘導M1 表型的過程中扮演重要角色。雖然過往研究已廣泛探討細胞外鈣離子攝取與內質網釋放之機制，但內溶小體(endolysosome)作為細胞內最主要的酸性鈣離子儲庫其內的鈣離子在巨噬細胞極化中是否存在關聯則相對未明。因此，本研究聚焦於兩種內溶小體陽離子通道--TPC2與TRPML2，這兩者主要表現在單核吞噬細胞系統中。我們以THP-1衍生之巨噬細胞作為模型，系統性地分析其向M1型極化過程中的變化，並探討內溶小體離子通道在抗白色念珠菌(Candida albicans)免疫中的角色。
首先，我們觀察到THP-1細胞自圓形的M0巨噬細胞轉變為具胞質延伸不規則形狀的M1巨噬細胞。接著我們發現，在極化早期(LPS與IFN-γ處理6小時)，TRPML2而非TPC2出現短暫性表現上升。另外，我們也透過內溶小體膜片鉗技術(endolysosomal patch-clamp)記錄證實，M0與M1巨噬細胞中皆具功能性TPC2電流，與基因表現結果一致。我們進一步以鈣指示劑(Oregon Green 488 Dye)測定內溶小體相對鈣含量後發現，M1巨噬細胞的鈣含量明顯高於M0，此現象在野生型(WT)和TPC2敲除細胞中都可以觀察到，然而在TRPML2敲除細胞中此差異消失。此外，使用TRPML2激動劑ML2-SA2處理後可提升細胞內鈣含量，而抑制劑ML-SI3處理則未顯著改變鈣含量。其次，在極化過程中加TPC2或TRPML2的專一性激動劑以增加通道活性，不論是在低濃度(1 μM)或是較高濃度(10 μM)的刺激下，都並未提升TNF-α與IL-6的表現，這些結果說明單純透過這些通道釋放鈣離子不足以驅動促發炎細胞激素的轉錄表現。最後，我們發現TRPML2的表現量在野生型及ece1Δ突變株的白色念珠菌感染中均於感染後期顯著上升，顯示TRPML2可能參與抗真菌免疫反應。總結而言，本研究揭示了TRPML2在調控內溶小體鈣動態與抗真菌免疫中的潛在功能，並強調其作為調節巨噬細胞功能狀態的重要性。 | zh_TW |
| dc.description.abstract | Macrophages, particularly the M1 phenotype, are essential for maintaining homeostasis and host defense during pathogen infection. Calcium signaling plays a pivotal role in macrophage polarization, especially in the transition to the M1 phenotype. While previous studies have extensively explored extracellular calcium uptake and calcium release from the endoplasmic reticulum (ER), the role of lysosomes, as primary intracellular acidic calcium reservoirs, remains poorly understood. In this study, we focus on two endolysosomal cation channels, TPC2 and TRPML2, which are predominantly expressed in the mononuclear phagocyte system. Using THP-1-derived macrophage as a model, we aim to perform a systematic analysis of macrophage polarization to M1 phenotype and investigate the role of endolysosomal ion channel in antifungal immunity against Candida albicans. First, we observed that THP-1 cells underwent a morphological change from rounded M0 to irregular M1 macrophages. We also demonstrated that TRPML2, but not TPC2, was transiently upregulated at the early stage of M1 polarization (LPS and IFN-γ treated for 6 hours). Patch-clamp recordings further confirmed the functional presence of TPC2-like currents in both M0 and M1 macrophages, consistent with gene expression data. Notably, we revealed that relative calcium content in endolysosome was significantly elevated in M1 macrophages compared to M0, a difference abolished in TRPML2 knockout cells but preserved in TPC2 knockouts. In addition, pharmacological activation of TRPML2 with ML2-SA2 elevated calcium content, whereas its inhibition with ML-SI3 showed no significant difference. Secondary, our results showed that increasing TPC2 or TRPML2 channel activity by applying channel-specific agonists during polarization failed to enhance the expression of TNF-α and IL-6, suggesting that calcium efflux via these channels alone is insufficient to drive polarization in term of pro-inflammatory cytokine transcription. Third, we found that TRPML2 expression was significantly induced upon both WT and ece1Δ mutant strain Candida albicans prolonged infection, indicating that TRPML2 play a potential role in response to antifungal immunity. To sum up, these findings highlight a previously underappreciated role of TRPML2 in calcium regulation and antifungal immunity and suggest its potential as a modulator of macrophage functional states. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2026-03-13T16:43:15Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2026-03-13T16:43:15Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員審定書 i
致謝 ii 中文摘要 iii Abstract v Figure content xii Table content xiv Supplemental information content xv Chapter 1. Introduction 1 1.1. Macrophage 1 1.1.1. THP-1 (Human acute monocytic leukemia cell line) 1 1.1.2. Macrophage differentiation and polarization 2 1.2. Macrophage and calcium 3 1.3. Candida albicans 5 1.3.1. Candida albicans and intracellular calcium in macrophage 6 1.4. Endolysosomal ion channel 7 1.4.1. TRPML2 and its role in immunity 8 1.4.2. TPC2 and its role in immunity 8 1.4.3. Agonists and antagonists 9 Chapter 2. Rationale and Specific Aims 11 2.1. Rationale 11 2.2. Specific aims 11 2.2.1. Characterizing Morphological and Molecular Differences between Polarized and Non-Polarized Macrophages through Endolysosomal Calcium Signaling Pathways 11 2.2.2. To explore the effects of endolysosomal Ca2+ channel-specific agonists on macrophage polarization efficiency 12 2.2.3. Investigating TRPML2 Expression and Function in Macrophage Responses to Candida albicans 13 Chapter 3. Materials and Methods 14 3.1. Cell and cell culture 14 3.2. Fungal and Fungal preparation 15 3.3. Drugs 15 3.4. Macrophage polarization 16 3.5. Cell viability assay 16 3.5.1 Cell viability assay with or without PMA resting 17 3.5.2. CCK-8 Cell Viability Assay Under Drug Treatment 18 3.6. RNA isolation and Reverse transcription 19 3.7 Quantitative Real-time-PCR (qPCR) 20 3.8 Oregon green 488 BAPTA-1 dextran (OG-BAPTA-dextran) imaging 21 3.9. Whole-endolysosomal patch-clamp 21 3.10. Statistics 23 3.11. Funding 23 Chapter 4. Results 24 4.1. The morphology changed between different stages of macrophages 24 4.2. Comparative analysis of rested and non-rested THP-1 macrophages in terms of morphology, viability, and TPC2/TRPML2 expression 24 4.3 Successful M1 polarization using LPS and IFN-γ was confirmed by mRNA expression of pro-inflammatory cytokines TNF-α and IL-6 compared to unstimulated M0 controls 26 4.4. Endolysosomal ion channel TPC2 and TRPML2 expression varied between different stages of macrophages 27 4.5. The knockout of TPC2 or TRPML2 did not affect the expression levels of each other. 28 4.6. The TPC2-like currents in M0 and M1 macrophages, consistent with the qPCR results, showed no significant differences. 29 4.7. The relative calcium content in M1 macrophages was significantly higher compared to that in M0 macrophages. 29 4.8. TRPML2 activity treated with channel-specific agonist or antagonist regulated relative calcium content in M0 and M1 macrophages 31 4.9. Evaluate the impact of adding additional channel-specific agonist during macrophage polarization on polarization efficiency 32 4.9.1. Channel-specific agonists TPC2-A1-N, TPC2-A1-P, and ML2-SA1 exhibited no significant cytotoxicity at a concentration of 1 μM after 24 hours of incubation 32 4.9.2. Applying channel-specific agonists to increase channel activity during the polarization process did not further enhance M1 polarization efficiency in terms of pro-inflammatory cytokine transcription 33 4.10. The expression levels of TRPML2 increased 24 hours post-infection with WT Candida albicans 34 4.11. The expression levels of TRPML2 increased after prolonged ece1Δ Candida albicans infection 35 Chapter 5. Discussion & Conclusion 37 Reference 44 | - |
| dc.language.iso | en | - |
| dc.subject | TPC2 | - |
| dc.subject | TRPML2 | - |
| dc.subject | 內溶小體離子通道 | - |
| dc.subject | 鈣離子 | - |
| dc.subject | 巨噬細胞M1極化 | - |
| dc.subject | 白色念珠菌 | - |
| dc.subject | TPC2 | - |
| dc.subject | TRPML2 | - |
| dc.subject | endolysosomal ion channel | - |
| dc.subject | calcium | - |
| dc.subject | macrophage M1 polarization | - |
| dc.subject | Candida albicans | - |
| dc.title | 探討內溶小體離子通道TRPML2在巨噬細胞M1極化與白色念珠菌的交互作用 | zh_TW |
| dc.title | Exploring the Role of Endolysosomal Ion Channel TRPML2 in Macrophage M1 Polarization and Its Interaction with Candida albicans | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 114-1 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 楊雅倩;蔡雨寰 | zh_TW |
| dc.contributor.oralexamcommittee | Ya-Chien Yang;Yu-Huan Tsai | en |
| dc.subject.keyword | TPC2,TRPML2內溶小體離子通道鈣離子巨噬細胞M1極化白色念珠菌 | zh_TW |
| dc.subject.keyword | TPC2,TRPML2endolysosomal ion channelcalciummacrophage M1 polarizationCandida albicans | en |
| dc.relation.page | 84 | - |
| dc.identifier.doi | 10.6342/NTU202504484 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2025-09-19 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學系 | - |
| dc.date.embargo-lift | 2030-09-14 | - |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-114-1.pdf 未授權公開取用 | 2.23 MB | Adobe PDF | 檢視/開啟 |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。