本文關(guān)鍵詞：TLR2與TLR4配體預(yù)處理對角膜基質(zhì)細(xì)胞抗煙曲霉菌炎癥反應(yīng)的影響，由筆耕文化傳播整理發(fā)布。

        研究背景：真菌性角膜炎是一種致盲率極高的感染性角膜病變,我國屬與該疾病高發(fā)區(qū)域,相關(guān)的臨床流行病學(xué)調(diào)查顯示其發(fā)病率逐年上升,因此,防治真菌性角膜炎成為我國致盲性眼科疾病的重要臨床課題。真菌性角膜炎致盲的重要原因之一就是真菌感染后,固有免疫所致的過度炎癥反應(yīng)。隨著研究的深入,Toll樣受體家族在角膜防御真菌感染過程中發(fā)揮的重要作用逐漸為人發(fā)現(xiàn)并重視。TLR2在與TLR4在抗真菌免疫應(yīng)答中占有著重要地位,TLR2可識別酵母聚糖,TLR4可識別脂多糖。隨后的研究指出以低濃度TLRs配體預(yù)處理誘導(dǎo)細(xì)胞,可以使其對再次“致死性”刺激反應(yīng)性下降和炎性細(xì)胞因子分泌減少,該現(xiàn)象曾被稱為"TLR耐受”。TLR預(yù)處理抑制多形核白細(xì)胞過度炎癥反應(yīng)的研究已有較多報道,但其在角膜細(xì)胞抗過度炎癥反應(yīng)的研究中較少,不同TLR配體協(xié)同耐受的研究更是鮮有報道。目的：研究酵母聚糖(Zymosan,TLR2特異性配體)單獨預(yù)處理或與脂多糖(LPS,TLR4特異性配體)聯(lián)合預(yù)處理,對角膜基質(zhì)細(xì)胞抗煙曲霉菌炎癥反應(yīng)的影響。方法：培養(yǎng)人永生化角膜基質(zhì)纖維細(xì)胞(telomerase-immortatized humancornea stroma fibroblast,THSF),以4×105個細(xì)胞/孔的濃度接種于6孔板,以不同濃度Zymosan(104ng/ml.10.ng/ml.100ng/ml.10ng/m1)預(yù)處理角膜基質(zhì)細(xì)胞12h,然后給予大劑量煙曲霉菌刺激4h,收取細(xì)胞,通過RT-PCR分析比較不同濃度Zymosan預(yù)處理對角膜基質(zhì)細(xì)胞炎性因子(TNF-α,IL-6)表達(dá)的影響。以4×105個細(xì)胞/孔的濃度將永生化角膜基質(zhì)細(xì)胞接種于6孔板,給與不同濃度Zymosan與LPS(103ng/ml Zymosan+lOng/ml LPS、100ng/ml Zymosan+10ng/mlLPS)聯(lián)合預(yù)處理,分別在預(yù)處理6h,12h,18h,24h,30h后,給予大劑量煙曲霉菌刺激4h,收取6孔板中的細(xì)胞上清,通過RT-PCR檢測角膜基質(zhì)細(xì)胞炎性因子(TNF-a,IL-6和IL-8)基因水平表達(dá)；收取基質(zhì)細(xì)胞,通過ELISA檢測培養(yǎng)液上清中炎性因子蛋白的分泌量,分析Zymosan與LPS聯(lián)合預(yù)處理對角膜基質(zhì)細(xì)胞炎癥反應(yīng)的影響。采用SPSS13.0統(tǒng)計軟件對數(shù)據(jù)分析,以均數(shù)±標(biāo)準(zhǔn)差表示,采用獨立樣本t檢驗與ANOVA分析比較各組間差異,P<0.05認(rèn)為差異有統(tǒng)計學(xué)意義。結(jié)果：1.1000ng/mlZymosan最大程度的抑制了TNF-a和1L-6的分泌。在一定濃度范圍內(nèi),Zymosan預(yù)處理濃度愈高,對角膜基質(zhì)炎性細(xì)胞因子分泌的抑制愈強。2.100ng/mlZymosan+10ng/ml LPS聯(lián)合預(yù)處理較二者單獨預(yù)處理更有效地抑制炎癥因子的分泌。結(jié)論：適宜濃度的TLR2配體的預(yù)處理可以抑制煙曲霉菌刺激導(dǎo)致的角膜基質(zhì)細(xì)胞炎癥因子的過度分泌,適宜濃度的TLR2與TLR4特異配體聯(lián)合預(yù)處理可以更有效地抑制炎性因子的過度分泌。

    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與TLR4配體預(yù)處理對角膜基質(zhì)細(xì)胞抗煙曲霉菌炎癥反應(yīng)的影響

目錄4-5CONTENTS5-6中文摘要6-8ABSTRACT8-9符號說明10-11前言11-13材料和方法13-22結(jié)果22-24討論24-27結(jié)論27-28附圖28-32參考文獻32-36綜述36-47    參考文獻42-47致謝47-48攻讀學(xué)位期間發(fā)表的論文48-49學(xué)位論文評閱及答辯情況表49

  本文關(guān)鍵詞：TLR2與TLR4配體預(yù)處理對角膜基質(zhì)細(xì)胞抗煙曲霉菌炎癥反應(yīng)的影響，，由筆耕文化傳播整理發(fā)布。