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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 廖泰慶 | zh_TW |
| dc.contributor.advisor | Albert Taiching Liao | en |
| dc.contributor.author | 劉芷均 | zh_TW |
| dc.contributor.author | Chih-Chun Liu | en |
| dc.date.accessioned | 2025-02-26T16:23:55Z | - |
| dc.date.available | 2025-02-27 | - |
| dc.date.copyright | 2025-02-26 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-02-10 | - |
| dc.identifier.citation | Aguilera, R., Saffie, C., Tittarelli, A., González, F. E., Ramírez, M., Reyes, D., Pereda, C., Hevia, D., García, T., Salazar, L., Ferreira, A., Hermoso, M., Mendoza-Naranjo, A., Ferrada, C., Garrido, P., López, M. N., & Salazar-Onfray, F. (2011). Heat-Shock Induction of Tumor-Derived Danger Signals Mediates Rapid Monocyte Differentiation into Clinically Effective Dendritic Cells. Clinical Cancer Research, 17(8), 2474–2483.
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Lactobacillus johnsonii activates porcine monocyte derived dendritic cells maturation to modulate Th cellular immune response. Cytokine, 144, 155581. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/97089 | - |
| dc.description.abstract | 樹突細胞（DC）是最強大的抗原呈現細胞，對於啟動T細胞依賴性免疫反應十分重要，可作為癌症免疫療法的重要工具。其中，透過DC與自體腫瘤細胞融合生成樹突細胞/腫瘤融合細胞是一種有效的癌症免疫療法策略。然而，這個方法在應用上受到癌症患犬可取得的腫瘤細胞數量可能不足，以及細胞潛在抗藥性細菌污染風險的限制。本研究的目的為利用不同的TLR配體誘導犬單核球來源樹突細胞（cMoDC）成熟，進一步表徵成熟樹突細胞（mDC）後，篩選出較適合誘導DC成熟的TLR配體，以犬黑色素瘤細胞株取代患犬的自體腫瘤細胞，並將其與mDC融合後，評估樹突細胞/腫瘤細胞株融合細胞後續活化免疫反應的潛力。我們從犬周邊血液單核細胞培養出未成熟樹突細胞（iDC），且將iDC分別以TLR3配體聚肌胞苷酸（Poly I:C）、TLR4配體脂多醣（LPS）及兩者併用誘導成熟後，以即時定量聚合酶連鎖反應（RT-qPCR）及流式細胞儀分析mDC的成熟度，再以細胞學試驗了解樹突細胞/腫瘤融合細胞的增生情形及誘導異體淋巴細胞增殖情形。結果顯示，不同TLR配體都能誘導iDC產生顯著的形態學變化，其中以Poly I:C誘導的mDC（mDC/P）呈現較粗壯的突起，以LPS誘導的mDC（mDC/L）則表現多個細長的針狀突起，而兩者併用誘導的mDC （mDC/PL）結合了兩者的特徵。mDC成熟度評估結果顯示，成熟標記CD83、共刺激分子CD80、CD86、以及細胞激素IL-12在所有組別中均上調，顯示各種刺激方法均能誘導成熟。另外CCR7在所有組別中均上調，而IFN-γ僅在mDC/L中上調，TNF-α在mDC/PL和mDC/L中上調。而在免疫抑制型細胞激素中，TGF-β在mDC/P和mDC/PL中下調，而IL-10在所有組別中均上調，其中mDC/PL上調最高。在所有組別中，mDC/L因其整體表現較優，被選為用來生成融合細胞的對象。後續細胞學試驗進一步表明mDC/L與腫瘤融合細胞在不顯示任何增殖的情況下仍保持活性，且能有效刺激淋巴細胞增殖。本研究證明，TLR3或TLR4配體都能誘導cMoDC來源iDC成熟，且LPS似乎是讓iDC成熟的較佳選擇。此外，融合細胞在安全性與刺激異體淋巴細胞增殖的能力表現良好，代表腫瘤細胞株有取代自體腫瘤細胞的潛力，未來可望應用在樹突細胞/腫瘤融合細胞的製備，為犬隻的癌症免疫治療開創新的可能性。 | zh_TW |
| dc.description.abstract | Dendritic cells (DCs) are the most powerful antigen-presenting cells and are crucial in activating T-cell-dependent immune responses. They can be an essential tool forcancer immunotherapy. Among the strategies developed, fusing DCs and autologous tumor cells to generate a DC/tumor hybrid is an effective approach for cancer immunotherapy. However, this strategy is limited by the potential insufficiency of tumor cells obtainable from cancer dogs and the risks of drug-resistant bacterial contamination during cell preparation. This study aimed to evaluate the capability of different Toll-like receptor (TLR) ligands in inducing the maturation of canine monocyte-derived dendritic cells (cMoDCs), characterize the resulting mature dendritic cells (mDCs), identify more effective TLR ligands for inducing maturation, and fuse mDCs with melanoma cell lines instead of primary tumor cells to evaluate the potential of DC/tumor fusion cells in activating immune responses. Immature dendritic cells (iDCs) were cultured from peripheral blood mononuclear cells of dogs and induced to mature using either the TLR3 ligand polyinosinic-polycytidylic acid (Poly I:C), the TLR4 ligand lipopolysaccharide (LPS), or a combination of both. Flow cytometry and RT-qPCR were used to analyze the maturity of the resulting mDCs, then cellular studies were conducted to evaluate their safety, viability and ability to activate allogeneic lymphocytes. Results showed that different TLR ligands induced significant morphological changes in iDCs. mDCs induced by Poly I:C (mDC/P) exhibited strong projections, while those induced by LPS (mDC/L) showed needle-like extensions. mDCs induced by the combination of Poly I:C and LPS (mDC/PL) exhibited the features of both. The analysis of mDCs' maturity revealed upregulation of maturation markers CD83, co-stimulatory molecules CD80 and CD86, and pro-inflammatory cytokine IL-12 in all groups, indicating successful maturation with all stimulation methods. Additionally, CCR7 was upregulated in all groups, IFN-γ was upregulated only in mDC/L, and TNF-α was upregulated in mDC/PL and mDC/L. For immunosuppressive cytokines, TGF-β was downregulated in mDC/P and mDC/PL, while IL-10 was upregulated in all groups, with the highest level observed in mDC/PL. Among the groups, mDC/L demonstrated superior overall performance and was selected for generating fusion cells. Consequent cell studies showed that mDC/L fused with tumor cells remained viable without proliferation and effectively stimulated lymphocyte proliferation. This study demonstrated that both TLR3 or TLR4 ligands can effectively induce the maturation of canine iDCs, with LPS being the more practical choice for DC maturation. In addition, viability assays ensured the safety of the mDC/tumor cell line hybrid for future immunotherapy. Furthermore, proliferation assays demonstrated that these hybrids can induce lymphocyte proliferation, emphasizing the capability of tumor cell lines to replace autologous tumor cells. These findings suggest that this approach could be applied to the preparation of DC/tumor fusion cells, highlighting their potential for future therapeutic applications in dog cancer. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-02-26T16:23:55Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-02-26T16:23:55Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 謝辭 I
摘要 II Abstract IV Contents VII Chapter 1 Literature Review 1 1.1 Introduction of dendritic cells 1 1.1.1 Biological characteristics and function 1 1.1.2 Subsets of DCs 1 1.1.3 Distribution and trafficking 3 1.2 Dendritic Cells in Cancer Immunity 4 1.2.1 Dendritic Cells and Tumor Microenvironment 4 1.2.2 Immunosuppressive mechanisms in cancer 5 1.3 Dendritic Cells Maturation and Lymphocyte Activation 5 1.3.1 Signal integration model of dendritic cell activation 6 1.3.2 PRRs and Pathogen-associated Molecular Patterns 7 1.4 T-cell activation signals 9 1.4.1 Signal 1: Antigen Presentation 10 1.4.2 Signal 2: Co-stimulation 10 1.4.3 Signal 3: Cytokine Signaling 10 1.4.4 Immune Memory Formation 12 1.5 DC-based immunotherapy 13 1.5.1 Strategies to Load Tumor Antigens 14 1.5.2 Fusion Hybrid Strategies 15 1.6 Summary 17 Chapter 2 Introduction 18 Chapter 3 Materials and Methods 21 3.1 Generation of canine monocyte-derived dendritic cells 21 3.1.1 PBMC isolation from PRBC 21 3.1.2 PBMC isolation from whole blood 22 3.1.3 Canine MoDC cultivation 22 3.2 Flow cytometry 23 3.3 Scanning electron microscope (SEM) 23 3.4 RNA extraction 24 3.5 Reverse transcription polymerase chain reaction (RT-PCR) 25 3.6 Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) 26 3.7 Fusion hybrid generation 27 3.8 Fusion hybrid safety assay 27 3.9 Lymphocyte proliferation assay 28 3.10 Statistical analysis 29 Chapter 4 Results 30 4.1 Generation of MoDCs 30 4.1.1 Yield efficiency comparison between PBMCs and PRBCs 30 4.1.2 Morphological changes from PBMCs to iDCs 30 4.1.3 Detection of TLR3 and TLR4 in iDCs 31 4.2 Maturation evaluation of canine MoDCs 31 4.2.1 Analysis by flow cytometry 31 4.2.2 Analysis by RT-qPCR 32 4.3 Fusion hybrids of mDC/melanoma cell line 34 4.4 Fusion hybrids safety assay 35 4.5 Lymphocyte proliferation assay 36 Chapter 5 Discussion 37 Tables 45 Table 1. Aim of DC activities 45 Table 2. List of primers used in this study 46 Table 3. Yield Efficiency Comparison 48 Table 4. Summary of DC characterization 49 Figures 50 Figure 1. Workflow for fusion hybrid production 50 Figure 2. Experimental design 51 Figure 3. Isolation of PBMCs using PRBCs and whole blood 52 Figure 4. Morphological changes from PBMCs to iDCs 53 Figure 5. RT-PCR analysis of TLR3 and TLR4 mRNA expression in canine iDC. 54 Figure 6. Morphological of MoDCs under different stimulation conditions. 55 Figure 7. Scanning electron microscopy analysis of cMoDCs 56 Figure 8. CD80 expression in MoDCs by flow cytometry 57 Figure 9. Surface markers mRNA expression in MoDCs by qRT-PCR 58 Figure 10. Co-stimulatory molecules mRNA expression in MoDCs by qRT-PCR 59 Figure 11. Pro-inflammatory cytokine mRNA expression in cMoDCs by qRT-PCR 60 Figure 12. Inhibitory cytokines mRNA expression in cMoDCs by qPCR 61 Figure 13. Morphological Characterization of DC-Melanoma Fusion Process 62 Figure 14. Cell proliferation and viability assessment of fusion hybrids 63 Figure 15. Proliferation of peripheral lymphocyte stimulated by fusion hybrid. 64 References 65 | - |
| dc.language.iso | en | - |
| dc.subject | 樹突細胞/腫瘤融合細胞 | zh_TW |
| dc.subject | 癌症免疫療法 | zh_TW |
| dc.subject | 犬單核球來源樹突細胞 | zh_TW |
| dc.subject | 犬黑色素瘤 | zh_TW |
| dc.subject | 類鐸受體配體 | zh_TW |
| dc.subject | 聚肌胞苷酸 | zh_TW |
| dc.subject | 脂多醣 | zh_TW |
| dc.subject | TLR ligands | en |
| dc.subject | Poly I:C | en |
| dc.subject | LPS | en |
| dc.subject | DC/tumor fusion hybrid | en |
| dc.subject | Canine melanoma | en |
| dc.subject | Cancer immunotherapy | en |
| dc.subject | Canine monocyte-derived dendritic cell | en |
| dc.title | 犬單核球來源樹突細胞/腫瘤融合細胞之功能性評估 | zh_TW |
| dc.title | Functional Evaluation of Canine Monocyte-Derived Dendritic Cell/Tumor Cell Line Fusion Hybrid | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-1 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 李繼忠;詹昆衛;林辰栖 | zh_TW |
| dc.contributor.oralexamcommittee | Jih-Jong Lee;Kun-Wei Chan;Chen-Si Lin | en |
| dc.subject.keyword | 癌症免疫療法,犬單核球來源樹突細胞,犬黑色素瘤,類鐸受體配體,聚肌胞苷酸,脂多醣,樹突細胞/腫瘤融合細胞, | zh_TW |
| dc.subject.keyword | Cancer immunotherapy,Canine monocyte-derived dendritic cell,Canine melanoma,TLR ligands,Poly I:C,LPS,DC/tumor fusion hybrid, | en |
| dc.relation.page | 74 | - |
| dc.identifier.doi | 10.6342/NTU202500560 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2025-02-11 | - |
| dc.contributor.author-college | 生物資源暨農學院 | - |
| dc.contributor.author-dept | 獸醫學系 | - |
| dc.date.embargo-lift | N/A | - |
| 顯示於系所單位： | 獸醫學系 | |
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