TLR2与TLR4配体预处理对角膜基质细胞抗烟曲霉菌炎症反应的影响

  本文关键词：TLR2与TLR4配体预处理对角膜基质细胞抗烟曲霉菌炎症反应的影响，由笔耕文化传播整理发布。

        研究背景：真菌性角膜炎是一种致盲率极高的感染性角膜病变,我国属与该疾病高发区域,相关的临床流行病学调查显示其发病率逐年上升,因此,防治真菌性角膜炎成为我国致盲性眼科疾病的重要临床课题。真菌性角膜炎致盲的重要原因之一就是真菌感染后,固有免疫所致的过度炎症反应。随着研究的深入,Toll样受体家族在角膜防御真菌感染过程中发挥的重要作用逐渐为人发现并重视。TLR2在与TLR4在抗真菌免疫应答中占有着重要地位,TLR2可识别酵母聚糖,TLR4可识别脂多糖。随后的研究指出以低浓度TLRs配体预处理诱导细胞,可以使其对再次“致死性”刺激反应性下降和炎性细胞因子分泌减少,该现象曾被称为"TLR耐受”。TLR预处理抑制多形核白细胞过度炎症反应的研究已有较多报道,但其在角膜细胞抗过度炎症反应的研究中较少,不同TLR配体协同耐受的研究更是鲜有报道。目的：研究酵母聚糖(Zymosan,TLR2特异性配体)单独预处理或与脂多糖(LPS,TLR4特异性配体)联合预处理,对角膜基质细胞抗烟曲霉菌炎症反应的影响。方法：培养人永生化角膜基质纤维细胞(telomerase-immortatized humancornea stroma fibroblast,THSF),以4×105个细胞/孔的浓度接种于6孔板,以不同浓度Zymosan(104ng/ml.10.ng/ml.100ng/ml.10ng/m1)预处理角膜基质细胞12h,然后给予大剂量烟曲霉菌刺激4h,收取细胞,通过RT-PCR分析比较不同浓度Zymosan预处理对角膜基质细胞炎性因子(TNF-α,IL-6)表达的影响。以4×105个细胞/孔的浓度将永生化角膜基质细胞接种于6孔板,给与不同浓度Zymosan与LPS(103ng/ml Zymosan+lOng/ml LPS、100ng/ml Zymosan+10ng/mlLPS)联合预处理,分别在预处理6h,12h,18h,24h,30h后,给予大剂量烟曲霉菌刺激4h,收取6孔板中的细胞上清,通过RT-PCR检测角膜基质细胞炎性因子(TNF-a,IL-6和IL-8)基因水平表达；收取基质细胞,通过ELISA检测培养液上清中炎性因子蛋白的分泌量,分析Zymosan与LPS联合预处理对角膜基质细胞炎症反应的影响。采用SPSS13.0统计软件对数据分析,以均数±标准差表示,采用独立样本t检验与ANOVA分析比较各组间差异,P<0.05认为差异有统计学意义。结果：1.1000ng/mlZymosan最大程度的抑制了TNF-a和1L-6的分泌。在一定浓度范围内,Zymosan预处理浓度愈高,对角膜基质炎性细胞因子分泌的抑制愈强。2.100ng/mlZymosan+10ng/ml LPS联合预处理较二者单独预处理更有效地抑制炎症因子的分泌。结论：适宜浓度的TLR2配体的预处理可以抑制烟曲霉菌刺激导致的角膜基质细胞炎症因子的过度分泌,适宜浓度的TLR2与TLR4特异配体联合预处理可以更有效地抑制炎性因子的过度分泌。

    Background:Fungal keratitis is a kind of infective corneal disease caused high-rate of blindness. China is an area with a high incidence of the disease. Relevant clinical epidemiology investigation showed that the incidence of fungal keratitis was rising year by year, therefore, prevention and treatment of fungal keratitis has become an important clinical subject of eye disease caused blindness in China.One of the important reasons of blindness caused by fungal keratitis is excessive inflammatory reaction in innate immunity to fungal infection. TLRs play an important role in immune response of cornea to fungi. TLR2recognizes its ligand as zymosan and TLR4recognizes lipopolysaccharide. Studies showed that pretreatment with low concentration of TLRs ligand induced low-reaction to the "deadly" stimulation and low-secretion of inflammatory cytokines of host, the phenomenon was called "TLRs tolerance". Reports showed that TLRs pretreatment inhibited excessive inflammatory reaction of PMNs. Studies about tolerance induced with with TLRs ligand pretreatment in corneal fibroblasts challenged with Aspergillus fumigatus were few, even less about synergy of different TLRs in tolerance.Purpose:To study the innate immunity in telomerase-immortalized human stroma fibroblasts (THSFs) challenged with Aspergillus fumigatus hyphae after co-pretreatment with Zymosan(TLR2special ligand) and LPS(TLR4special ligand). Methods:THSFs were cultured in6-well plates at a density of4×105cells per well, and pretreated with different concentration of zymosan(104ng/mh103ng/m、100ng/ml、10ng/ml) for4hours, then challenged with high dose of Aspergillus fumigatus hyphae4hours. The expression of TNF-a and IL-6was detected by RT-PCR. THSFs were pretreated with the best concentration of zymosan and/or lipopolysaccharide (LPS) at different time periods (6h,12h,18h,24h or30h) and then re-stimulated with A. fumigatus hyphae. The expression of inflammatory cytokines (TNF-a, IL-6and IL-8) detected by RT-PCR and ELISA. Co-pretreated THSFs with the differrnt concentration of zymosan and LPS (103ng/ml Zymosan+10ng/ml LPS、100ng/ml Zymosan+10ng/ml LPS) for various periods (6h,12h,18h,24h and30h), then stimulated with A. Fumigatus hyphae for4h. The culture media and cells were harvested at the indicated time-points for measurement of cytokines (TNF-a, IL-6and IL-8) mRNA and protein levels. Statistical analysis was determined by one-way analysis of variance (ANOVA) and Student’s t test using SPSS (version13.0). Differences were considered statistically significant at P<0.05.Results:1. Pretreatment with10000ng/ml Zymosan led to maximum suppression. In the certain concentration range, pretreatment of THSFs with Zym suppressed gene expression of inflammatory cytokines (TNF-a and IL-6).2. Co-pretreatment with10000ng/ml Zym and10ng/ml LPS suppressed gene expression and protein secretion more strongly compared with pretreatment with zymosan or LPS alone.Conclusions:Pretreatment of THSFs with TLR2specific ligand, zymosan resulted in a state of A. fumigatus hyphae tolerance. Co-pretreatment with TLR2and4ligands (zymosan and LPS) led to a more strongly state of A. fumigatus hyphae tolerance.

TLR2与TLR4配体预处理对角膜基质细胞抗烟曲霉菌炎症反应的影响

目录4-5CONTENTS5-6中文摘要6-8ABSTRACT8-9符号说明10-11前言11-13材料和方法13-22结果22-24讨论24-27结论27-28附图28-32参考文献32-36综述36-47    参考文献42-47致谢47-48攻读学位期间发表的论文48-49学位论文评阅及答辩情况表49

  本文关键词：TLR2与TLR4配体预处理对角膜基质细胞抗烟曲霉菌炎症反应的影响，，由笔耕文化传播整理发布。