中文摘要：

目的探讨人角膜上皮组织和细胞系（THCE）对Toll样受体（TLR）2和TLR4的表达及后者介导THCE对烟曲霉菌（AF）抗原炎性反应的影响。方法用免疫印迹（Western blot）和免疫细胞化学方法检测人角膜上皮组织和细胞系THCE的TLR2和TLR4蛋白质表达与分布；采用自制的AF菌丝体片段（5×10^6／mL）和培养上清提取物（牛血清白蛋白当量浓度为10μg／ml）抗原，刺激培养的THCE细胞，于刺激1、2、4及8h收集培养上清，用酶联免疫吸附试验（EHSA）方法检测白细胞介素（IL）8和肿瘤坏死因子（TNF）α的水平。于刺激后30min、1及2h收集细胞，Western blot检测IKBα的表达变化以评价核转录因子kB的活化；采用抗体封闭实验分析封闭TLR2和TLR4对THCE细胞表达IL-8和TNF—α的影响。结果Western blot和免疫细胞化学结果显示人角膜上皮组织和THCE细胞均表达TLR2和TLR4蛋白质；AF菌丝体或培养上清抗原刺激THCE细胞后，培养上清液中IL-8和TNF-α于1h后开始升高，至8h分别达到（64．71±5．15）pg／ml和（32．46±3．28）pg／ml（菌丝体刺激组）及（94．94±11．92）pg／ml和（48．70±3．32）pg／ml（上清刺激组），分别为对照组THCE细胞培养上清中IL-8和TNF—α浓度的3．0倍和2．5倍及4．5倍和3．5倍（均P〈0．01）；同时Western blot检测AF菌丝体和AF上清刺激30min后THCE细胞的IKBα表达（平均灰度值）分别由对照组的51．57±5．58和49．23±3．49下降为10．31±1．30和8．15±2．37（均P〈0．01），2h后恢复至对照组水平。在AF菌丝体刺激组，单纯封闭TLR2或TLR4部分抑制THCE细胞IL-8和TNF-α的分泌（均P〈0．05），同时封闭两种受体则明显抑制了IL-8和TNF-α分泌（均P〈0．01）；AF上清抗原刺激组，单纯封闭TLR4和联合封闭两个受体均可明显抑制IL．8和TNF—α的分泌（均P〈0．01），而单纯封闭TLR2未明显抑制二者的分泌（均P〉0．05）?

英文摘要：

Objective To determine Toll-like receptors （TLR）2 and TLR4 expression in human corneal epithelial tissue and cell line （THCE）, and its activation by aspergillus fumigatus （AF） in inflammatory response. Methods The expression of TLR2 and TLR4 protein in human corneal epithelial tissue and THCE was detected by Western blot and immunocytochemistry. THCE was challenged with AF mycelium fragment （ 5 × 10^6/ml ） and supernatant extract agent （ equivalent to bovine serum albumin 10 μg/ml）. IL-8 and TNF-α in THCE supernatant were detected by ELISA at 1, 2, 4 and 8 h post stimulation. The protein of IKBα in THCE cells was assayed by Western blot at 30 min, 1 h and 2 h after treatment. Antibody blocking test was utilized to evaluate the effect on IL-8 and TNF-α expression of THCE by blocking TLR2 and（or） TLR4 before challenge with AF agent. Results TLR2 and TLR4 protein were expressed in human corneal epithelial tissue and THCE. The IL-8 and TNF-α level in THCE supernatant was elevated at 1 h, increased to （ 64. 71 ±5. 15 ） pg/ml and （ 32.46 ±3.28 ） pg/ml （ AF mycelium challenge group）, （94. 94 ±11.92 ） pg/ml and （ 48.70 ±3.32 ） pg/ml （ AF supernatant challenge group） 8 h postchallenged, which was 3.0 times and 2.5 times, 4.5 times and 3.5 times to that of control group respectively（P 〈0. 01）. The activity of IKBα in THCE cells was decreased to 10. 31 ±1.30 （gray scale value） and 8. 15 ± 2.37 at 30 min after challenged with AF mycelium or supernatant extract agent compared to 51.57 ±.58 and 49. 23 ±.49 of control group（P 〈0. 01 ）, and was reverted at 2 h. The secretion of IL-8 and TNF-α was partly inhibited by blocking TLR2 or TLR4（ P 〈0. 05 ）, obviously inhibited by blocking TLR2 and TLR4（50% and 40% compared to that of control group） （ P 〈0. 01 ） when challenged with AF mycelium. And that was markedly inhibited by blocking TLR4 or blocking TLR2 and TLR4 when challenged with AF supernatant（ P 〈0. 01 ）. The secretio