Li Jing, Shen JiangBo, Beuerman Roger W
Singapore Eye Research Institute, Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Mol Vis. 2007 Jun 8;13:813-22.
To determine the expression and function of toll-like receptors (TLRs) in human conjunctival, limbal and corneal epithelial cells.
Expression of TLRs was examined by real-time polymerase chain reaction, immunohistochemistry, and western blot analysis in human conjunctival, corneal and limbal epithelial cells and tissues. Ligand-stimulated nuclear factor kappaB activation; interleukin 6 and interleukin 8 protein secretion was measured in the cultured conjunctival and limbal epithelial cells by ELISA analysis.
Expression of TLR1, 2, 3, 5, and 6 was found in all conjunctival and limbal epithelial cell samples analyzed by real time PCR and western blot. TLR4 and TLR9 transcripts were undetectable in some samples by real-time PCR. TLR7, 8 and 10 transcripts were not detected by real time PCR in any of the samples tested. TLR1, 2, 3, 4, and 5 proteins were found in conjunctival, limbal and corneal epithelium by immunohistochemistry. Cultured conjunctival epithelial cells expressed significantly lower levels of TLRs than uncultured conjunctival cells obtained by applying nitrocellulose paper to the bulbar conjunctival surface. Cultured limbal and conjunctival cells responded to stimulation by polyriboinosinic polyribocytidylic acid (poly[I:C]), palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK) and flagellin with increased secretion of IL-6 and IL-8 and the activation of NFkappaB. Peptidoglycans (PGN) and CpG DNA caused increased NFkappaB activity; however, only conjunctival epithelial cells showed increased cytokine secretion. Lipoteichoic acid (LTA) or lipopolysacchride (LPS) did not change cytokine secretion or NFkappaB levels in either cell type.
The TLRs found in human conjunctival and limbal epithelial cells provide a basis for responses to many common ocular pathogens. Although the mRNA and protein for TLR4 and TLR2 was found, neither conjunctival or limbal cells in culture responded to LPS or LTA stimulation.
确定Toll样受体（TLRs）在人结膜、角膜缘和角膜上皮细胞中的表达及功能。
通过实时聚合酶链反应、免疫组织化学和蛋白质印迹分析检测人结膜、角膜和角膜缘上皮细胞及组织中TLRs的表达。采用酶联免疫吸附分析（ELISA）测定培养的结膜和角膜缘上皮细胞中配体刺激的核因子κB激活、白细胞介素6和白细胞介素8蛋白分泌情况。
通过实时PCR和蛋白质印迹分析发现，在所有分析的结膜和角膜缘上皮细胞样本中均有TLR1、2、3、5和6的表达。实时PCR在一些样本中未检测到TLR4和TLR9转录本。在任何测试样本中，实时PCR均未检测到TLR7、8和10转录本。免疫组织化学显示，结膜、角膜缘和角膜上皮中存在TLR1、2、3、4和5蛋白。与通过将硝酸纤维素纸贴于球结膜表面获取的未培养结膜细胞相比，培养的结膜上皮细胞表达的TLRs水平显著降低。培养的角膜缘和结膜细胞对聚肌苷酸-聚胞苷酸（poly[I:C]）、棕榈酰-3-半胱氨酸-丝氨酸-赖氨酸-4（Pam3CSK）和鞭毛蛋白刺激有反应，IL-6和IL-8分泌增加，核因子κB激活。肽聚糖（PGN）和CpG DNA导致核因子κB活性增加；然而，只有结膜上皮细胞显示细胞因子分泌增加。脂磷壁酸（LTA）或脂多糖（LPS）在两种细胞类型中均未改变细胞因子分泌或核因子κB水平。
人结膜和角膜缘上皮细胞中发现的TLRs为对许多常见眼部病原体的反应提供了基础。虽然发现了TLR4和TLR2的mRNA和蛋白，但培养的结膜或角膜缘细胞对LPS或LTA刺激均无反应。
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