Toll样受体在人眼角膜上皮和永生化人角膜上皮细胞系的表达及功能研究

The expression and functionality of Toll-like receptors in human corneal epithelium and tolerated human corneal epithelial cell line

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 functionality of Toll-like receptors （TLRs） 1-9 in human corneal epithelium and cell line THCE （tolerated human corneal epithelial）. Methods Reverse- transcriptase polymerase chain reaction （RT-PCR） analysis was used to determine the expression of TLR1-9 mRNA in the specimens of human corneal epithelial cells from 20 persons, 5 males and 10 females, aged 18-25, undergoing photorefractive keratectomy （ PRK）, and in cultured THCE cells. Human peripheral blood mononuclear cells （PBMCs） were used as positive control. The expression of TLR2 protein and TLR4 protein were detected with Western blotting. KEM-2 fluid containing anti-TLR4 antibody was used to block the THEC cells for 30 minutes, then KEM-2 fluid containing PolyI：C or LPS, ligands for TLR 3 and 4, was used to stimulate the THEC cells, 1,4, and 8 hours after the concentrations of IL-8 in the supernatant were determined by ELISA. Results The human corneal epithelial cells strongly expressed TLR1, 2, 3, 5, 6, and 9 mRNA, weakly expressed TLR7 and 8 mRNA, and very weakly expressed TLR4 mRNA. Negative expression of TLR3, 4, 6, and 8 mRNA was seen in 1 case, a female aged 22. Weak expression of TLR5 mRNA was seen in 1 case, a female aged 20. The THCE cells showed the same expression pattern as the healthy human corneal epithelial cells, except for the strong expression of TLRg. The human PBMCs, as positive controls, strongly expressed TLR1 - 4 and 8 mRNA, weakly expressed TLRS, 6, and 9 mRNA, and very weakly expressed TLR7 mRNA. The human corneal epithelial cells, cultured THEC cells, and PBMCs all expressed TLR2 and 4 proteins. The concentrations of IL-8 in the supernatant of the culture fluid of THEC cells increased along with the time of stimulation of LPS and Poly：C, ligands for TLR3 and 4, and reached to 297.33 pg/ml and 229.67 pg/ml respectively 8 hours after, 10 times and 7 times those of the control group （ both P 〈 0.05 ）. 30 minutes after the blocking of TLR4 with anti-TLR4 antibody LPS was used to stimulate the THEC, the concentration of IL-8 was 88.54 pg/ml, being 30% that of the un-blocked group （P 〈 0.05）. Conclusion Human corneal epithelium expresses TLR1 - 9 at different levels. THCE cell line is an excellent line to study TLRs function.