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研究生(外文):Ho Thi Hang
論文名稱(外文):Expression profile, subcellular localization and signaling pathway analysis of fish-specific toll-like receptor 25 (TLR25) in Nile tilapia (Oreochromis niloticus)
指導教授(外文):Po-Tsang Lee
口試委員(外文):Chiu-Ming WenCheng-Hui LinPo-Tsang Lee
外文關鍵詞:Nile tilapiaTLRinnate immunitygene expressionAeromonasStreptococcuscytokineinterferonpattern recognition protein
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尼羅魚 (Oreochromis niloticus) 是熱帶和亞熱帶地區生產主要的食用魚類之一,但是,該行業一直面臨受到微生物感染的重大挑戰。了解宿主如何啟動針對入侵之微生物的免疫反應是解決疾病爆發和對抗疾病的首要條件。類toll受體在演化上為保守的蛋白質家族,其可以辨識病原體相關的分子模式,因此,它們在先天免疫中扮演著非常重要的角色。TLR25是TLR1亞家族中一個新發現的魚類特異性成員。本研究透過TLR25組織表現模式、受細菌和TLR配體刺激後之基因表現量調控、在人類和魚類細胞中TLR25的蛋白定位及TLR25可能誘導的訊號途徑研究尼羅魚TLR25 (OnTLR25)的分子和功能特徵。在免疫相關器官 (如脾臟和頭腎)中,OnTLR25的轉錄表現較高,而在受到細菌攻擊後會提升其表現量。此外,結果顯示OnTLR25位於質體轉染之吳郭魚頭腎 (THK)細胞株中的胞內。此外,在THK細胞中,大量表現缺少配體辨識區域之OnTLR25可誘導促發炎細胞因子的基因表現,如腫瘤壞死因子α、介白素 (IL-1β)、IL-8、IL-12a和干擾素-d2.13。綜合以上結果,TLR25可能在尼羅魚先天免疫系統的抗菌反應中扮演著重要的角色。
The Nile tilapia (Oreochromis niloticus) is one of the major food fish species produced in tropical and subtropical regions. However, this industry has been facing significant challenges from microbial infections. Understanding how hosts initiate immune responses against invading microbes is the first requirement for addressing disease outbreak prevention and disease resistance. Toll-like receptors (TLRs) are a family of evolutionarily conserved proteins that can recognize pathogen-associated molecular patterns (PAMPs). They thus play an essential role in innate immunity. TLR25 is a newly identified fish-specific member of the TLR1 subfamily. In this study, we investigate the molecular and functional characteristics of O. niloticus TLR25 (OnTLR25) via tissue expression patterns, gene expression modulation after challenge with bacteria and TLR ligands, subcellular localization in human and fish cells, and the signaling pathways TLR25 may induce. Transcriptional levels of OnTLR25 are high in immune-related organs such as the spleen and head kidney, and are increased following bacterial challenges. In addition, we show that OnTLR25 preferentially localizes to the intracellular compartment in transfected tilapia head kidney (THK) cell line. Furthermore, overexpression of the truncated form of OnTLR25 in THK cell line induced the expression of proinflammatory cytokines, such as tumor necrosis factor α, interleukin (IL)-1β, IL-8, IL-12a, and interferon-d2.13. Combined, our results suggest that TLR25 is likely to play an important role in the antimicrobial responses of the innate immune system of Nile tilapia.
中文摘要 IV
2.1. Geographic distribution and tilapia production 4
2.1.1. Geographic distribution 4
2.1.2. Tilapia production 4
2.2. Disease problem in tilapia 5
2.2.1. Bacterial disease 6
2.2.2. Viral disease 8
2.2.3. Parasitic disease 9
2.3. Innate immune system 10
2.4. Pattern recognition receptors (PRRs) 11
2.4.1. NOD-like receptors (NLRs) 12
2.4.2. C-type lectin receptors (CLRs) 13
2.4.3. RIG-1 like receptors (RLRs) 14
2.4.4. Toll-like receptors (TLRs) 14 Discovery of toll-like receptors 14 Structure of toll-like receptors 15 TLR localization 17 TLR signaling pathways 18 MyD88-dependent signaling pathway 18 TRIF-dependent signaling pathway 19 Ligands 20
2.4.5. Effectors in the TLR signaling pathways 21 Antimicrobial peptides (AMPs) 22 Type I Interferons (IFNs) 22 Cytokines 23 TNFα 24 Interleukin-1β (IL-1β) 25 Interleukin-12 (IL-12) 26 Interferon-gamma (IFNγ) 26 Chemokines and interleukin-8 (IL-8) 27
2.4.6. Toll-like receptor 25 (TLR25) 28
2.5. Lymphoid organs 29
2.5.1. Thymus 29
2.5.2. Kidney 29
2.5.3. Spleen 30
3.1. Experimental fish, infection, and tissues sampling 31
3.2. Gene cloning and plasmid construction 31
3.3. Bioinformatics analyses 33
3.4. Cell culture 34
3.5. Stimulation of THK cells 34
3.6. Total RNA extraction, cDNA synthesis, and quantitative real-time polymerase chain reaction (qRT-PCR) assay 34
3.7. Western blot analysis 36
3.8. Confocal microscopy 37
3.9. Luciferase assay 37
3.10. Activation of OnTLR25 signaling pathways in THK cells 38
3.11. Statistical analysis 38
4.1. Cloning, phylogenetic analysis, and sequence comparison 39
4.2. Domain organization and genome synteny analysis 40
4.3. Expression of OnTLR25 in healthy fish tissues 41
4.4. Expression modulation of OnTLR25 in ligand-stimulated THK cells 42
4.5. Expression of OnTLR25 in Nile tilapia challenged with Streptococcus agalactiae, Aeromonas hydrophila, and poly I:C 42
4.6. Cellular localization of Nile tilapia TLR25 43
4.7. The truncated form of OnTLR25 activates expression of proinflammatory cytokines and type I IFN in THK cells 43
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