Research Center for Inflammation and Regenerative Medicine, Faculty of Life and Medical Sciences, Doshisha University, Doshisha, Kyoto, Japan.
Br J Ophthalmol. 2010 Nov;94(11):1528-32. doi: 10.1136/bjo.2010.180554. Epub 2010 Jul 23.
The authors previously reported that human ocular surface epithelium expressed TLR3 and that its ligand polyI:C stimulated the secretion of IL-6, IL-8 and IFN-β. In this study, to examine comprehensive effects of polyI:C stimulation of primary human conjunctival epithelial cells (PHCjECs), the authors performed a gene-expression analysis of the polyI:C-stimulated PHCjECs using oligonucleotide microarrays, GeneChip.
The transcripts upregulated upon polyI:C stimulation in PHCjECs from two individuals were examined using GeneChip. Eleven new upregulated transcripts of interest were confirmed by quantitative real-time PCR (RT-PCR), and seven proteins produced by those transcripts were examined by ELISA or immunoblot analysis in PHCjECs from three other individuals, respectively.
GeneChip analysis showed that 150 transcripts were upregulated more than threefold and that 47 transcripts were upregulated more than 10-fold upon polyI:C stimulation in the PHCjECs. Eleven of the 47 upregulated transcripts (CXCL11, RIG-I, IL28A, CXCL10, CCL5, CCL4, MDA5, IL7R, TSLP, CCL20 and ICAM-1) were significantly upregulated upon polyI:C stimulation by quantitative RT-PCR, and the levels of seven proteins of the transcripts CXCL11, CXCL10, CCL5, CCL20, TSLP, RIG-I and MDA5 were confirmed by ELISA or immunoblot analysis to increase significantly in polyI:C-stimulated PHCjECs.
Our results might show that TLR3 of conjunctival epithelium could not only induce antiviral innate immune responses but also regulate the allergic reactions.
作者先前报道，人眼表面上皮细胞表达 TLR3，其配体 polyI:C 可刺激 IL-6、IL-8 和 IFN-β 的分泌。在这项研究中，为了研究 polyI:C 刺激原代人结膜上皮细胞（PHCjEC）的综合作用，作者使用寡核苷酸微阵列（GeneChip）对 polyI:C 刺激的 PHCjEC 进行了基因表达分析。
使用 GeneChip 检测了 2 个人的 PHCjEC 中 polyI:C 刺激后的上调转录本。通过定量实时 PCR（qRT-PCR）验证了 11 个新的上调转录本，并在另外 3 个人的 PHCjEC 中分别通过 ELISA 或免疫印迹分析检测了这些转录本产生的 7 种蛋白质。
GeneChip 分析显示，在 PHCjEC 中，polyI:C 刺激后有 150 个转录本上调超过 3 倍，有 47 个转录本上调超过 10 倍。在定量 RT-PCR 中，这 47 个上调转录本中有 11 个（CXCL11、RIG-I、IL28A、CXCL10、CCL5、CCL4、MDA5、IL7R、TSLP、CCL20 和 ICAM-1）在 polyI:C 刺激下显著上调，通过 ELISA 或免疫印迹分析，CXCL11、CXCL10、CCL5、CCL20、TSLP、RIG-I 和 MDA5 这 7 种转录本的蛋白水平在 polyI:C 刺激的 PHCjEC 中也明显增加。
我们的结果可能表明结膜上皮细胞的 TLR3 不仅能诱导抗病毒先天免疫反应，还能调节过敏反应。
