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目的探讨Toll样受体(TLR)1～9在人眼角膜上皮和上皮细胞系的表达及其功能性。方法以健康人外周血单个核细胞(PBMC)为阳性对照,收集20例健康青年人角膜上皮标本和培养永生化人角膜上皮细胞系(THCE)细胞,用半定量反转录聚合酶链反应检测TLR1～9mRNA表达;Western印迹检测TLR2、4的蛋白质表达;TLR3和TLR4的配体对THCE刺激后酶联免疫检测分泌IL8的变化,结合抗体封闭实验,研究角膜上皮TLR的功能性。结果与PBMC比较,人角膜上皮强表达TLR1、2、3、5、6、9,弱表达TLR8,微弱表达TLR4;20例角膜上皮标本中发现1例TLR3、4、6、8阴性表达,1例TLR5微弱表达;人角膜上皮在蛋白质水平表达TLR2、4;THCE细胞与人角膜上皮有相似的TLR表达谱;LPS和PolyI:C刺激THCE1、4、8h后IL8分泌增加,8h时分别达到对照组的10倍和7倍(均P<0.05),抗体封闭TLR4可以阻断LPS诱导的IL8分泌。结论人角膜上皮表达TLR1～9,但不同TLRs表达水平有差异。THCE是研究人角膜上皮TLR表达和功能的良好细胞系。
Objective To investigate the expression and function of Toll-like receptors (TLRs) 1-9 in human corneal epithelial cells and epithelial cell lines. Methods Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were used as positive control. Twenty healthy young human corneal epithelial samples were collected and cultured for immortalized human corneal epithelial cell line (THCE). Semi-quantitative reverse transcriptase-polymerase chain reaction The expression of TLR1 ~ 9 mRNA was detected by Western blotting. The TLR3 and TLR4 ligands were detected by enzyme-linked immunosorbent assay (ELISA) to detect the changes of IL8 secreted by THCE. Antibody blocking experiments were performed to investigate the function of TLR in corneal epithelium. Results Compared with PBMC, human corneal epithelium strongly expressed TLR1, 2, 3, 5, 6, 9, weakly expressed TLR8 and weakly expressed TLR4. One case of TLR3, 4, 6, and 8 negative expression was found in 20 corneal epithelial samples TLR5 was weakly expressed in human corneal epithelium; TLR2,4 was expressed at the protein level in human corneal epithelium; TLR expression was similar in THCE cells and human corneal epithelium; IL8 secretion was increased 4, 8 and 8 h after LPS and PolyI: C stimulation, 10-fold and 7-fold higher (all P <0.05). Antibody-blocking TLR4 blocked LPS-induced IL8 secretion. Conclusion Human corneal epithelium expressed TLR1 ~ 9, but different TLRs expression levels were different. THCE is a well-established cell line that studies human corneal epithelial TLR expression and function.