Toll样受体及其信号通路在角膜真菌感染中的作用
|
摘要
第一部分：
Toll样受体在人眼角膜上皮细胞的表达和功能研究
目的 探讨Toll样受体(Toll-like receptors，TLRs)1～TLR9在人眼角膜上皮及角膜上皮细胞系的表达及其功能。
材料和方法 用半定量RT-PCR方法检测20例健康青年人角膜上皮组织标本、永生化人眼角膜上皮细胞系(THCE)细胞及健康人外周血单个核细胞(PBMCs)的TLR1～TLR9 mRNA表达；免疫印记(Western blot)和免疫细胞化学方法检测人角膜上皮组织和角膜上皮细胞系THCE的TLR2和TLR4蛋白质表达及分布；用TLR3的配体聚肌胞苷酸(PolyI：C，1000g／ml)和TLR4的配体绿脓杆菌脂多糖(LPS，100ng／ml)刺激THCE细胞，于刺激后1h，2h，4h，8h分别收集细胞培养上清，用酶联免疫吸附试验(ELISA)检测其IL-8的变化。结合TLR4抗体封闭实验，研究TLRs在人眼角膜上皮细胞的表达及其功能。
结果 20例角膜上皮组织的RT-PCR结果显示：TLR1-TLR3，TLR5，TLR6及TLR9在人眼角膜上皮细胞为强表达，TLR7和TLR8弱表达，TLR4微弱表达。20例标本中1例TLR3，TLR4，TLR6及TLR8为阴性表达，1例TLR5微弱表达。THCE细胞与人眼角膜上皮组织有相似的TLRs表达谱；Western blot及免疫细胞化学结果显示人眼角膜上皮组织及THCE在蛋白质水平表达TLR2及TLR4；刺激实验显示LPS和PolyI：C刺激THCE后其培养上清中的IL-8浓度明显上升，8h时分别达到对照组的10倍(P＜0.05)和7倍(P＜0.05)。抗体封闭TLR4明
Objective To investigate the expression and functionality of Toll-like receptor (TLR)1-TLR9 in human corneal epithelium and its cell line tolerated human corneal epithelial(THCE).
Material and methods Expression of TLR1-TLR9 mRNA was determined by reverse- transcriptase polymerase chain reaction (RT-PCR) analysis of 20 human corneal epithelial cells separated by photorefractive keratectomy (PRK), and THCE cell line. Human peripheral blood mononuclear cells (PBMCs) were used as positive control. Expressions of TLR2 and TLR4 protein were detected with Western blot and immunocytochemistry. Functional relevance of TLR was tested by enzyme linked immunsorbent assay (ELISA) analysis of IL-8 production after stimulating cell lines with ligands for TLR3 or TLR4(Poly I:C or LPS). The effect of anti-TLR4 antibody on LPS-induced IL-8 production of THCE was observed.
Results TLR1-TLR9 mRNA species were detected in both human corneal epithelial cells and THCE cells. Human corneal epithelium cells strongly express
Toll样受体在人眼角膜上皮细胞的表达和功能研究
目的 探讨Toll样受体(Toll-like receptors，TLRs)1～TLR9在人眼角膜上皮及角膜上皮细胞系的表达及其功能。
材料和方法 用半定量RT-PCR方法检测20例健康青年人角膜上皮组织标本、永生化人眼角膜上皮细胞系(THCE)细胞及健康人外周血单个核细胞(PBMCs)的TLR1～TLR9 mRNA表达；免疫印记(Western blot)和免疫细胞化学方法检测人角膜上皮组织和角膜上皮细胞系THCE的TLR2和TLR4蛋白质表达及分布；用TLR3的配体聚肌胞苷酸(PolyI：C，1000g／ml)和TLR4的配体绿脓杆菌脂多糖(LPS，100ng／ml)刺激THCE细胞，于刺激后1h，2h，4h，8h分别收集细胞培养上清，用酶联免疫吸附试验(ELISA)检测其IL-8的变化。结合TLR4抗体封闭实验，研究TLRs在人眼角膜上皮细胞的表达及其功能。
结果 20例角膜上皮组织的RT-PCR结果显示：TLR1-TLR3，TLR5，TLR6及TLR9在人眼角膜上皮细胞为强表达，TLR7和TLR8弱表达，TLR4微弱表达。20例标本中1例TLR3，TLR4，TLR6及TLR8为阴性表达，1例TLR5微弱表达。THCE细胞与人眼角膜上皮组织有相似的TLRs表达谱；Western blot及免疫细胞化学结果显示人眼角膜上皮组织及THCE在蛋白质水平表达TLR2及TLR4；刺激实验显示LPS和PolyI：C刺激THCE后其培养上清中的IL-8浓度明显上升，8h时分别达到对照组的10倍(P＜0.05)和7倍(P＜0.05)。抗体封闭TLR4明
Objective To investigate the expression and functionality of Toll-like receptor (TLR)1-TLR9 in human corneal epithelium and its cell line tolerated human corneal epithelial(THCE).
Material and methods Expression of TLR1-TLR9 mRNA was determined by reverse- transcriptase polymerase chain reaction (RT-PCR) analysis of 20 human corneal epithelial cells separated by photorefractive keratectomy (PRK), and THCE cell line. Human peripheral blood mononuclear cells (PBMCs) were used as positive control. Expressions of TLR2 and TLR4 protein were detected with Western blot and immunocytochemistry. Functional relevance of TLR was tested by enzyme linked immunsorbent assay (ELISA) analysis of IL-8 production after stimulating cell lines with ligands for TLR3 or TLR4(Poly I:C or LPS). The effect of anti-TLR4 antibody on LPS-induced IL-8 production of THCE was observed.
Results TLR1-TLR9 mRNA species were detected in both human corneal epithelial cells and THCE cells. Human corneal epithelium cells strongly express
引文
1. Lemaitre B, Nicolas E, Michaut L, et al. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell, 1996,86:973-983.
2. Takeda K, Akira S. TLR signaling pathway. Semin Immunol, 2004,16 :3~9.
3. Zhang D, Zhang G, Matthew S, et al. A Toll-like receptor that prevents infection by uropathogenic bacteria. Science, 2004, 303:1522-1526.
4. Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett, 2003, 85: 85-95.
5. Mambula SS, Sau K, Henneke P, et al. Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus. J Biol Chem. 2002,277: 39320-39326.
6. Kariko K, Ni H, Capodici J, et al. mRNA is an endogenous ligand for Toll-like receptor 3. J Biol Chem. 2004,279:12542-12550.
7. Lund JM, Alexopoulou L, Sato A, et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. PNAS. 2004,101: 5598-5603.
8. Heil F, Hemmi H, Hochrein H, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science. 2004,303:1526-1529.
9. O'Neill L. After the toll rush. Science. 2004,303:1481-1482.
10. Karin M , Yamamoto Y, Wang QM, et al. The IKK NF-kB system: A treasure trove for drug development. Nature Reviews/Drug discovry, 2004,3:17-26.
11. Song PI, Abrabam TA, Park Y, et al. The expression of functional LPS receptor proteins CD14 and toll-like receptor 4 in human corneal cells. Invest Opthalmol Vis Sci. 2001,42: 2867-2877.
12. Hise AG, Gillette-Ferguson I, Pearlman E. Immunopathogenesis of Onchocerca volvulus keratitis (river blindness): a novel role for TLR4 and endosymbiotic Wolbachia bacteria. J Endotoxin Res. 2003,9:390-394.
13. Zhang J, Xu K, Ambati B, et al. Toll-like receptor 5-mediated corneal epithelial inflammatory responses to Pseudomonas aeruginosa flagellin. Invest Ophthamol Vis Sci. 2003, 44: 4247-4254.
14. Wang JE, Warris A, Ellingsen EA, et al. Involvement of CD14 and Toll-like receptors in activation of human monocytes by Aspergillus fumigatus hyphae. Infect Immun, 2001, 69: 2402-2406.
15. Netea MG, de Graaf C, Vonk A, et al. The role of Toll-like receptors in the defense against disseminated candidiasis. J Infect Dis, 2002,185:1483-1489.
16. Young SL, Lyddon TD, Jorgenson RL, et al. Expression of Toll-like receptors in human endometrial epithelial cells and cell lines. Am J Reprod Immunol, 2004, 52: 67-73.
17. Hawn TR, Verbon A, Lettinga KD, et al. A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to legionnaires' disease. J Exp Med, 2003,198:1563-1572.
18. Pruett SB, Fan R, Zheng Q. Acute ethanol administration profoundly alters poly I:C-induced cytokine expression in mice by a mechanism that is not dependent on corticosterone. Life Sciences, 2003,72:1825-1839.
19. Dcushima H, Nishida T, Takeda K, et al. Expression of Toll-like receptors 2 and 4 is downregulated after operation. Surgery, 2004,135: 376-385.
20. Manigold T, Bocker U, Hanck C, et al. Differential expression of toll-like receptors 2 and 4 in patients with liver cirrhosis. Eur J Gastroenterol Hepatol, 2003, 15: 275-282.
21. Renshaw M, Rockwell J, Engleman C, et al. Impaired Toll-like receptor expression and function in aging. J Immunol, 2002,169:4697-4701.
1．王丽娅，张月琴，王印其，等．中国三地区真菌性角膜病致病菌种的调查．中华眼科杂志，2000，36：138-139．
2．谢立信，史伟云，董晓光，等．108例真菌性角膜炎的临床和组织病理学研究．眼科研究，1999，17：283-285．
3. Braedel S, Radsak M, Einsele H, et al. Aspergillus fumigatus antigens activate innate immune cells via toll-like receptors 2 and 4. Br J Haematol, 2004, 125: 392-399.
4. Takeda K, Akira S. TLR signaling pathway. Semin Immunol, 2004, 16: 3-9.
5. Zhang D, Zhang G, Matthew S, et al. A toll-like receptor that prevents infection by uropathogenic bacteria. Science, 2004, 303: 1522-1526.
6. Meier A, Kirschning, C. J, Nikolaus T, et al. Toll-like receptor (TLR) 2 and TLR4 are essential for Aspergillus-induced activation of routine macrophages. Cell Microbiol, 2003, 5: 561-570.
7. Song PI, Abrabam TA, Park Y, et al. The expression of functional LPS receptor proteins CD14 and Toll-like receptor 4 in human corneal cells. Invest Opthalmol Vis Sci, 2001, 42: 2867-2877.
8．范延红，赵连友，郑强荪，等。核因子κB在精氨酸血管升压素诱导人鼠心肌成纤维细胞一氧化氮合成中的作用 生理学报，2003，55：417—422．
9. Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett, 2003, 85: 85-95.
10. Mambula SS, Sau K, Henneke P, et al. Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus. J Biol Chem, 2002, 277: 39320-39326.
11. Young SL, Lyddon TD, Jorgenson RL, et al. Expression of toll-like receptors in human endometrial epithelial cells and cell lines. Am J Reprod Immunol, 2004, 52: 67-73.
12. O'Neill L. After the toll rush. Science, 2004, 303: 1481-1482.
13. Zhang J, Xu K, Ambati B, et al. Toll-like receptor 5-mediated corneal epithelial inflammatory responses to Pseudomonas aeruginosa flagellin. Invest Ophthamol Vis Sci, 2003, 44: 4247-4254.
14. Netea MG, Warris A, Meer J, et al. Aspergillus fumigatus evades immune recognition during germination through loss of Toll-like receptor-4-mediated signal transduction. J Infect Dis, 2003, 188: 320-326.
15. Hemmi H, Kaisho T, Takeuchi O, et al. Small antiviral compounds activate immune cells via TLR7 MyD88-dependent signalling pathway. Nat Immunol. 2002, 3: 196-200.
16. Kawai T, Adachi O, Ogawa T, et al. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity. 1999, 11: 115-122.
17. Yamamoto M, Sato S, Hemmi H, et al. Essential role of TIRAP/Mal for activation of the signaling cascade shared by TLR2 and TLR4. Nature. 2002, 420: 324-329.
18. Yamamoto M, Sato S, Hemmi H, et al. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science. 2003, 301: 640-643.
19. Sharma S, Oever BR, Grandvaux N, et al. Triggering the interferon antiviral response through an IKK-related pathway. Science. 2003, 300: 1148-1151.
20. Horng T, Barton GM, Flavell RA, et al. The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature. 2002, 420: 329-333.
21. Karin M, Yamamoto Y, Wang M. The IKK NF-κB system: a treasure trove for drug development. Nat Rev Drug discov. 2004, 3: 17-25.
22. Viriyakosol S, Fierer J, Brown GD, et al. Innate immunity to the pathogenic fungus Coccidioides posadasii is dependent on Toll-like receptor 2 and Dectin-1. Infect Immun, 2005, 73; 1553-1560.
23. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, et al. Recognition of Commensal Microflora by Toll-Like Receptors Is Required for Intestinal Homeostasis. 2004, Cell, 118; 229-241.
24. Hasan UA, Trinchieri G, Vlach J. TLR signaling stimulates cell cycle entry and progression in fibroblasts. J Biol Chem, 2005, 280; 20620-20627.
25．邹雄，张利宁主编。分子免疫学与临床。济南，山东科技出版社，2003年5 月第一版：155—160。
26. Park E, Jia J, Decreases MR. Taurine Chloramine Inhibits Lymphocyte Proliferation and Cytokine Production in Activated Human Leukocytes. Clinical Immunology, 2002, 102; 179-184.
27. Karin M, Yamamoto Y, Wang QM. The IKK NF-κB system: A treasure trove for drug development. Nature reviews/drug discovery, 2004, 3: 17-26.
1. Bellocchio S, Montagnoli C, Bozza S, et al. The contribution of the Toll-like/IL-1 receptor superfamily to innate and adaptive immunity to fungal pathogens in vivo. J Immunol, 2004, 172: 3059-3069.
2．王丽娅，张月琴，王印其，等．中国三地区真菌性角膜病致病菌种的调查．中华眼科杂志，2000，36：138-139．
3．谢立信，史伟云，董晓光，等．108例真菌性角膜炎的临床和组织病理学研究．眼科研究，1999，17：283-285．
4．谢立信，李绍伟，史伟云，等．共焦显微镜在真菌性角膜炎临床诊断中的应用．中华眼科杂志，1999，35：7-9．
5．陈瑞娥，戴文丽，孙志坚．真菌性角膜溃疡病原菌及其诱因分析．中华检验医学杂志，2002，25：364．
6. Behrens-Baumann W, Klinge B, Ruchel R. Topical fluconazole for experimental candida keratitis in rabbits. British Journal of Ophthalmology, 1990, 74: 40-42.
7. O'Day DM. Orally administered antifungal therapy for experimental keratomycosis. Trans Am Ophthalmol Soc, 1990, 88: 685-725.
8. O'Day DM. Studies in experimental keratomycosis. Curr Eye Res, 1985, 4: 243-52.
9. Braedel S, Radsak M, Einsele H, et al. Aspergillus fumigatus antigens activate innate immune cells via toll-like receptors 2 and 4. Br J Haematol, 2004, 125: 392-399.
10. Meier A, Kirschning, C. J, Nikolaus T, et al. Toll-like receptor (TLR) 2 and TLR4 are essential for Aspergillus-induced activation of murine macrophages. Cell Microbiol, 2003, 5: 561-570.
11. Netea MG, Van Der Graaf CA, Vonk AG, et al. The role of toll-like receptor (TLR) 2 and TLR4 in the host defense against disseminated candidiasis. J Infect Dis, 2002, 185: 1483-1489.
12. Mambula SS, Sau K, Henneke P, et al. Toll-like Receptor (TLR) Signaling in Response to Aspergillus fumigatus. J Biol Chem, 2002, 277: 39320-39326.
13. Shoham S, Huang C, Chen JM, et al. Toll-like receptor 4 mediates intracellular signaling without TNF-alpha release in response to Cryptococcus neoformans polysaccharide capsule, J Immunol, 2001, 166: 4620-4626.
14. Villamon E, Gozalbo D, Roig P, et al. Toll-like receptor-2 is essential in murine defenses against Candida albicans infections. Microbes Infect, 2004, 6: 1-7.
15. Netea MG, Sutmuller R, Hermann C, et al. Toll-like receptor 2 inhibits cellular responses against Candida albicans through pathways mediated by IL-10 and CD4+CD25+regulatory T cells. J Immunol, 2004, 172: 3712-3718.
16．高建鲁，吴欣怡．Toll样受体在人眼角膜上皮和永生化人角膜上皮细胞系的表达及功能研究．中华医学杂志，2005，85：2269-2273．
17．林学颜，张玲．细胞因子．见：林学颜，张玲，主编．现代细胞与分子免疫学．第一版．北京：科学出版社，2000．233-261．
18. Lemaitre B, Nicolas E, Michaut L, ct al. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell, 1996, 86: 973-983.
19. Takcda K, Akira S. TLR signaling pathway. Semin Immunol, 2004, 16: 3-9.
20. Zhang D, Zhang G, Matthew S, et al. A toll-like receptor that prevents infection by uropathogenic bacteria. Science, 2004, 303: 1522-1526.
21. Zhang J, Xu K, Ambati B, et al. Toll-like receptor 5-mediated corneal epithelial inflammatory responses to Pseudomonas aeruginosa flagellin. Invest Ophthamol Vis Sci, 2003, 44: 4247-4254.
22. Flo TH, Halaas O, Torp S, et al. Differential expression of Toll-like receptor 2 in human cells. J Leukoc Biol, 2001, 69: 474-481.
1. Lemaitre B, Nicolas E, Michaut L, et al. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell. 1996,86: 973-983.
2. Takeda K, Akira S. TLR signaling pathway. Semin Immunol. 2004,16: 3-9.
3. Zhang D, Zhang G, Matthew S, et al. A toll-like receptor that prevents infection by uropathogenic bacteria. Science. 2004, 303:1522-1526.
4. Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunology Letters. 2003, 85: 85-95.
5. Mambula SS, Sau K, Henneke P, et al. Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus. J Biol Chem. 2002,277: 39320-39326.
6. Kariko K, Ni H, Capodici J, et al. mRNA is an endogenous ligand for Toll-like receptor 3. J Biol Chem. 2004, 279:12542-12550.
7. Lund JM, Alexopoulou L, Sato A, et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. PNAS. 2004,101: 5598-5603.
8. Heil F, Hemmi H, Hochrein H, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science. 2004,303:1526-1529.
9. O'Neill L. After the toll rush. Science. 2004,303:1481-1482.
10. Schnare M, Holt AC, Takeda K, et al. Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein MyD88. Curr Biol. 2000,10: 1139-1142.
11. Hemmi H, Kaisho T, Takeuchi O, et al. Small antiviral compounds activate immune cells via TLR7 MyD88-dependent signalling pathway. Nat Immunol. 2002,3:196-200.
12. Kawai T, Adachi O, Ogawa T, et al. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity. 1999,11:115-122.
13. Yamamoto M, Sato S, Hemmi H, et al. Essential role of TIRAP/Mal for activation of the signaling cascade shared by TLR2 and TLR4. Nature. 2002,420:324-329.
14. Yamamoto M, Sato S, Hemmi H, et al. Role of adaptor TRIF in the MyD88- independent toll-like receptor signaling pathway. Science. 2003,301:640-643.
15. Sharma S, Oever BR, Grandvaux N, et al. Triggering the interferon antiviral response through an IKK-related pathway. Science. 2003, 300:1148-1151.
16. Horng T, Barton GM, Flavell RA, et al. The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature. 2002,420:329-333.
17. Karin M, Yamamoto Y, Wang M. The IKK NF-kB system: a treasure trove for drug development. Nat Rev Drug discov. 2004, 3:17-25.
18. Yoshida S, Ono M, Shono T. Involvement of interleukin-8, vascular endothelial growth factor and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis. Mol Cell Biol. 1997,17: 4015-4023.
19. Song PI, Abraham TA, Park Y, et al. The expression of functional LPS receptor proteins CD 14 and toll-like receptor 4 in human corneal cells. Invest Opthalmol Vis Sci. 2001,42: 2867-2877.
20. Hise AG, Gillette-Ferguson I, Pearlman E. Immunopathogenesis of Onchocerca volvulus keratitis (river blindness): a novel role for TLR4 and endosymbiotic Wolbachia bacteria. J Endotoxin Res. 2003,9:390-394.
21. Zhang J, Xu K, Ambati B, et al. Toll-like receptor 5-mediated corneal epithelial inflammatory responses to Pseudomonas aeruginosa flagellin. Invest Ophthamol Vis Sci. 2003,44: 4247-4254.
22. Ueta M, Nochi T, Jang MH, et al. Intracellularly expressed TLR2 and TLR4 contribution to an immunosilent environment at the ocular mucosal epithelium. J Immunol. 2004,173:3337-3347.
2. Takeda K, Akira S. TLR signaling pathway. Semin Immunol, 2004,16 :3~9.
3. Zhang D, Zhang G, Matthew S, et al. A Toll-like receptor that prevents infection by uropathogenic bacteria. Science, 2004, 303:1522-1526.
4. Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett, 2003, 85: 85-95.
5. Mambula SS, Sau K, Henneke P, et al. Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus. J Biol Chem. 2002,277: 39320-39326.
6. Kariko K, Ni H, Capodici J, et al. mRNA is an endogenous ligand for Toll-like receptor 3. J Biol Chem. 2004,279:12542-12550.
7. Lund JM, Alexopoulou L, Sato A, et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. PNAS. 2004,101: 5598-5603.
8. Heil F, Hemmi H, Hochrein H, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science. 2004,303:1526-1529.
9. O'Neill L. After the toll rush. Science. 2004,303:1481-1482.
10. Karin M , Yamamoto Y, Wang QM, et al. The IKK NF-kB system: A treasure trove for drug development. Nature Reviews/Drug discovry, 2004,3:17-26.
11. Song PI, Abrabam TA, Park Y, et al. The expression of functional LPS receptor proteins CD14 and toll-like receptor 4 in human corneal cells. Invest Opthalmol Vis Sci. 2001,42: 2867-2877.
12. Hise AG, Gillette-Ferguson I, Pearlman E. Immunopathogenesis of Onchocerca volvulus keratitis (river blindness): a novel role for TLR4 and endosymbiotic Wolbachia bacteria. J Endotoxin Res. 2003,9:390-394.
13. Zhang J, Xu K, Ambati B, et al. Toll-like receptor 5-mediated corneal epithelial inflammatory responses to Pseudomonas aeruginosa flagellin. Invest Ophthamol Vis Sci. 2003, 44: 4247-4254.
14. Wang JE, Warris A, Ellingsen EA, et al. Involvement of CD14 and Toll-like receptors in activation of human monocytes by Aspergillus fumigatus hyphae. Infect Immun, 2001, 69: 2402-2406.
15. Netea MG, de Graaf C, Vonk A, et al. The role of Toll-like receptors in the defense against disseminated candidiasis. J Infect Dis, 2002,185:1483-1489.
16. Young SL, Lyddon TD, Jorgenson RL, et al. Expression of Toll-like receptors in human endometrial epithelial cells and cell lines. Am J Reprod Immunol, 2004, 52: 67-73.
17. Hawn TR, Verbon A, Lettinga KD, et al. A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to legionnaires' disease. J Exp Med, 2003,198:1563-1572.
18. Pruett SB, Fan R, Zheng Q. Acute ethanol administration profoundly alters poly I:C-induced cytokine expression in mice by a mechanism that is not dependent on corticosterone. Life Sciences, 2003,72:1825-1839.
19. Dcushima H, Nishida T, Takeda K, et al. Expression of Toll-like receptors 2 and 4 is downregulated after operation. Surgery, 2004,135: 376-385.
20. Manigold T, Bocker U, Hanck C, et al. Differential expression of toll-like receptors 2 and 4 in patients with liver cirrhosis. Eur J Gastroenterol Hepatol, 2003, 15: 275-282.
21. Renshaw M, Rockwell J, Engleman C, et al. Impaired Toll-like receptor expression and function in aging. J Immunol, 2002,169:4697-4701.
1．王丽娅，张月琴，王印其，等．中国三地区真菌性角膜病致病菌种的调查．中华眼科杂志，2000，36：138-139．
2．谢立信，史伟云，董晓光，等．108例真菌性角膜炎的临床和组织病理学研究．眼科研究，1999，17：283-285．
3. Braedel S, Radsak M, Einsele H, et al. Aspergillus fumigatus antigens activate innate immune cells via toll-like receptors 2 and 4. Br J Haematol, 2004, 125: 392-399.
4. Takeda K, Akira S. TLR signaling pathway. Semin Immunol, 2004, 16: 3-9.
5. Zhang D, Zhang G, Matthew S, et al. A toll-like receptor that prevents infection by uropathogenic bacteria. Science, 2004, 303: 1522-1526.
6. Meier A, Kirschning, C. J, Nikolaus T, et al. Toll-like receptor (TLR) 2 and TLR4 are essential for Aspergillus-induced activation of routine macrophages. Cell Microbiol, 2003, 5: 561-570.
7. Song PI, Abrabam TA, Park Y, et al. The expression of functional LPS receptor proteins CD14 and Toll-like receptor 4 in human corneal cells. Invest Opthalmol Vis Sci, 2001, 42: 2867-2877.
8．范延红，赵连友，郑强荪，等。核因子κB在精氨酸血管升压素诱导人鼠心肌成纤维细胞一氧化氮合成中的作用 生理学报，2003，55：417—422．
9. Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett, 2003, 85: 85-95.
10. Mambula SS, Sau K, Henneke P, et al. Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus. J Biol Chem, 2002, 277: 39320-39326.
11. Young SL, Lyddon TD, Jorgenson RL, et al. Expression of toll-like receptors in human endometrial epithelial cells and cell lines. Am J Reprod Immunol, 2004, 52: 67-73.
12. O'Neill L. After the toll rush. Science, 2004, 303: 1481-1482.
13. Zhang J, Xu K, Ambati B, et al. Toll-like receptor 5-mediated corneal epithelial inflammatory responses to Pseudomonas aeruginosa flagellin. Invest Ophthamol Vis Sci, 2003, 44: 4247-4254.
14. Netea MG, Warris A, Meer J, et al. Aspergillus fumigatus evades immune recognition during germination through loss of Toll-like receptor-4-mediated signal transduction. J Infect Dis, 2003, 188: 320-326.
15. Hemmi H, Kaisho T, Takeuchi O, et al. Small antiviral compounds activate immune cells via TLR7 MyD88-dependent signalling pathway. Nat Immunol. 2002, 3: 196-200.
16. Kawai T, Adachi O, Ogawa T, et al. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity. 1999, 11: 115-122.
17. Yamamoto M, Sato S, Hemmi H, et al. Essential role of TIRAP/Mal for activation of the signaling cascade shared by TLR2 and TLR4. Nature. 2002, 420: 324-329.
18. Yamamoto M, Sato S, Hemmi H, et al. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science. 2003, 301: 640-643.
19. Sharma S, Oever BR, Grandvaux N, et al. Triggering the interferon antiviral response through an IKK-related pathway. Science. 2003, 300: 1148-1151.
20. Horng T, Barton GM, Flavell RA, et al. The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature. 2002, 420: 329-333.
21. Karin M, Yamamoto Y, Wang M. The IKK NF-κB system: a treasure trove for drug development. Nat Rev Drug discov. 2004, 3: 17-25.
22. Viriyakosol S, Fierer J, Brown GD, et al. Innate immunity to the pathogenic fungus Coccidioides posadasii is dependent on Toll-like receptor 2 and Dectin-1. Infect Immun, 2005, 73; 1553-1560.
23. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, et al. Recognition of Commensal Microflora by Toll-Like Receptors Is Required for Intestinal Homeostasis. 2004, Cell, 118; 229-241.
24. Hasan UA, Trinchieri G, Vlach J. TLR signaling stimulates cell cycle entry and progression in fibroblasts. J Biol Chem, 2005, 280; 20620-20627.
25．邹雄，张利宁主编。分子免疫学与临床。济南，山东科技出版社，2003年5 月第一版：155—160。
26. Park E, Jia J, Decreases MR. Taurine Chloramine Inhibits Lymphocyte Proliferation and Cytokine Production in Activated Human Leukocytes. Clinical Immunology, 2002, 102; 179-184.
27. Karin M, Yamamoto Y, Wang QM. The IKK NF-κB system: A treasure trove for drug development. Nature reviews/drug discovery, 2004, 3: 17-26.
1. Bellocchio S, Montagnoli C, Bozza S, et al. The contribution of the Toll-like/IL-1 receptor superfamily to innate and adaptive immunity to fungal pathogens in vivo. J Immunol, 2004, 172: 3059-3069.
2．王丽娅，张月琴，王印其，等．中国三地区真菌性角膜病致病菌种的调查．中华眼科杂志，2000，36：138-139．
3．谢立信，史伟云，董晓光，等．108例真菌性角膜炎的临床和组织病理学研究．眼科研究，1999，17：283-285．
4．谢立信，李绍伟，史伟云，等．共焦显微镜在真菌性角膜炎临床诊断中的应用．中华眼科杂志，1999，35：7-9．
5．陈瑞娥，戴文丽，孙志坚．真菌性角膜溃疡病原菌及其诱因分析．中华检验医学杂志，2002，25：364．
6. Behrens-Baumann W, Klinge B, Ruchel R. Topical fluconazole for experimental candida keratitis in rabbits. British Journal of Ophthalmology, 1990, 74: 40-42.
7. O'Day DM. Orally administered antifungal therapy for experimental keratomycosis. Trans Am Ophthalmol Soc, 1990, 88: 685-725.
8. O'Day DM. Studies in experimental keratomycosis. Curr Eye Res, 1985, 4: 243-52.
9. Braedel S, Radsak M, Einsele H, et al. Aspergillus fumigatus antigens activate innate immune cells via toll-like receptors 2 and 4. Br J Haematol, 2004, 125: 392-399.
10. Meier A, Kirschning, C. J, Nikolaus T, et al. Toll-like receptor (TLR) 2 and TLR4 are essential for Aspergillus-induced activation of murine macrophages. Cell Microbiol, 2003, 5: 561-570.
11. Netea MG, Van Der Graaf CA, Vonk AG, et al. The role of toll-like receptor (TLR) 2 and TLR4 in the host defense against disseminated candidiasis. J Infect Dis, 2002, 185: 1483-1489.
12. Mambula SS, Sau K, Henneke P, et al. Toll-like Receptor (TLR) Signaling in Response to Aspergillus fumigatus. J Biol Chem, 2002, 277: 39320-39326.
13. Shoham S, Huang C, Chen JM, et al. Toll-like receptor 4 mediates intracellular signaling without TNF-alpha release in response to Cryptococcus neoformans polysaccharide capsule, J Immunol, 2001, 166: 4620-4626.
14. Villamon E, Gozalbo D, Roig P, et al. Toll-like receptor-2 is essential in murine defenses against Candida albicans infections. Microbes Infect, 2004, 6: 1-7.
15. Netea MG, Sutmuller R, Hermann C, et al. Toll-like receptor 2 inhibits cellular responses against Candida albicans through pathways mediated by IL-10 and CD4+CD25+regulatory T cells. J Immunol, 2004, 172: 3712-3718.
16．高建鲁，吴欣怡．Toll样受体在人眼角膜上皮和永生化人角膜上皮细胞系的表达及功能研究．中华医学杂志，2005，85：2269-2273．
17．林学颜，张玲．细胞因子．见：林学颜，张玲，主编．现代细胞与分子免疫学．第一版．北京：科学出版社，2000．233-261．
18. Lemaitre B, Nicolas E, Michaut L, ct al. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell, 1996, 86: 973-983.
19. Takcda K, Akira S. TLR signaling pathway. Semin Immunol, 2004, 16: 3-9.
20. Zhang D, Zhang G, Matthew S, et al. A toll-like receptor that prevents infection by uropathogenic bacteria. Science, 2004, 303: 1522-1526.
21. Zhang J, Xu K, Ambati B, et al. Toll-like receptor 5-mediated corneal epithelial inflammatory responses to Pseudomonas aeruginosa flagellin. Invest Ophthamol Vis Sci, 2003, 44: 4247-4254.
22. Flo TH, Halaas O, Torp S, et al. Differential expression of Toll-like receptor 2 in human cells. J Leukoc Biol, 2001, 69: 474-481.
1. Lemaitre B, Nicolas E, Michaut L, et al. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell. 1996,86: 973-983.
2. Takeda K, Akira S. TLR signaling pathway. Semin Immunol. 2004,16: 3-9.
3. Zhang D, Zhang G, Matthew S, et al. A toll-like receptor that prevents infection by uropathogenic bacteria. Science. 2004, 303:1522-1526.
4. Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunology Letters. 2003, 85: 85-95.
5. Mambula SS, Sau K, Henneke P, et al. Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus. J Biol Chem. 2002,277: 39320-39326.
6. Kariko K, Ni H, Capodici J, et al. mRNA is an endogenous ligand for Toll-like receptor 3. J Biol Chem. 2004, 279:12542-12550.
7. Lund JM, Alexopoulou L, Sato A, et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. PNAS. 2004,101: 5598-5603.
8. Heil F, Hemmi H, Hochrein H, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science. 2004,303:1526-1529.
9. O'Neill L. After the toll rush. Science. 2004,303:1481-1482.
10. Schnare M, Holt AC, Takeda K, et al. Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein MyD88. Curr Biol. 2000,10: 1139-1142.
11. Hemmi H, Kaisho T, Takeuchi O, et al. Small antiviral compounds activate immune cells via TLR7 MyD88-dependent signalling pathway. Nat Immunol. 2002,3:196-200.
12. Kawai T, Adachi O, Ogawa T, et al. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity. 1999,11:115-122.
13. Yamamoto M, Sato S, Hemmi H, et al. Essential role of TIRAP/Mal for activation of the signaling cascade shared by TLR2 and TLR4. Nature. 2002,420:324-329.
14. Yamamoto M, Sato S, Hemmi H, et al. Role of adaptor TRIF in the MyD88- independent toll-like receptor signaling pathway. Science. 2003,301:640-643.
15. Sharma S, Oever BR, Grandvaux N, et al. Triggering the interferon antiviral response through an IKK-related pathway. Science. 2003, 300:1148-1151.
16. Horng T, Barton GM, Flavell RA, et al. The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature. 2002,420:329-333.
17. Karin M, Yamamoto Y, Wang M. The IKK NF-kB system: a treasure trove for drug development. Nat Rev Drug discov. 2004, 3:17-25.
18. Yoshida S, Ono M, Shono T. Involvement of interleukin-8, vascular endothelial growth factor and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis. Mol Cell Biol. 1997,17: 4015-4023.
19. Song PI, Abraham TA, Park Y, et al. The expression of functional LPS receptor proteins CD 14 and toll-like receptor 4 in human corneal cells. Invest Opthalmol Vis Sci. 2001,42: 2867-2877.
20. Hise AG, Gillette-Ferguson I, Pearlman E. Immunopathogenesis of Onchocerca volvulus keratitis (river blindness): a novel role for TLR4 and endosymbiotic Wolbachia bacteria. J Endotoxin Res. 2003,9:390-394.
21. Zhang J, Xu K, Ambati B, et al. Toll-like receptor 5-mediated corneal epithelial inflammatory responses to Pseudomonas aeruginosa flagellin. Invest Ophthamol Vis Sci. 2003,44: 4247-4254.
22. Ueta M, Nochi T, Jang MH, et al. Intracellularly expressed TLR2 and TLR4 contribution to an immunosilent environment at the ocular mucosal epithelium. J Immunol. 2004,173:3337-3347.