尘螨变应原rDer p1诱导HaCaT细胞表达胸腺基质淋巴生成素的研究

Effect of the house dust mite allergen rDer pl on the production of thymic stromal lymphopoietin by human keratinocytes

目的 探讨尘螨变应原rDer p1在体外对角质形成细胞表达胸腺基质淋巴生成素（TSLP）的影响。 方法 体外培养人角质形成细胞株HaCaT细胞，予以0.1、1和10 mg/L尘螨变应原rDer p1与蛋白酶活化受体2（PAR2）特异性激动剂SLGIKV（500 μmol/L）和拮抗剂VKGILS（500 μmol/L）共培养，以无血清培养基为空白对照。用ELISA法和荧光定量PCR法检测各实验组和对照组TSLP蛋白及mRNA表达水平；通过激光共聚焦显微镜检测细胞内钙流变化分析rDer p1诱导HaCaT细胞产生TSLP和PAR2受体活化的关系。 结果 1 mg/L和10 mg/L rDer p1组培养12 h上清中TSLP水平分别为（155.5 ± 5.9） ng/L和（228.8 ± 28.7） ng/L，显著高于空白对照组（54.3 ± 13.9 ng/L，P 〈 0.01），TSLP表达水平随rDer p1浓度增加而升高。SLGIKV组TSLP表达［（166.2 ± 8.8） ng/L］也显著高于空白对照组（P 〈 0.01）。10 mg/L rDer p1组和SLGIKV组TSLP mRNA相对表达量在8 h最高，分别为（3.28 ± 0.27）倍和（2.15 ± 0.26）倍，与4 h和24 h相比差异有统计学意义（P 〈 0.01）。SLGIKV可引起HaCaT细胞钙内流增加；10 mg/L rDer p1也可引起HaCaT细胞钙内流增加，经过PAR2受体特异性阻断剂VKGILS（500 μmol/L）处理后再给予rDer p1刺激，细胞内钙内流峰值明显降低。 结论 尘螨变应原rDer p1能够通过部分活化HaCaT细胞表面的PAR2受体诱导产生促炎细胞因子TSLP。

Objective To evaluate the in vitro effect of the house dust mite allergen rDer p1 on the production of thymic stromal lymphopoietin （TSLP） by human keratinocytes. Methods Human HaCaT keratinocytes were cultured in the presence of different concentrations （0.1, 1 and 10 mg/L） of rDer p1, SLGIKV （a specific agonist of protease-activated receptor-2, 500 μmol/L） and VKGILS （a specific antagonist of protease-activated receptor-2, 500 μmol/L） alone or in combination for different durations. Subsequently, enzyme-linked immunosorbent assay （ELISA） and fluorescence-based quantitative PCR were performed to detect the expression levels of TSLP protein in the culture supernatant of and TSLP mRNA in these cells respectively, confocal laser scanning microscopy was used to visualize intracellular calcium influx which reflected the activation of protease-activated receptor-2 （PAR2）. Those HaCaT cells cultured in serum-free medium served as the blank control group. Statistical analysis was done by t test and one-way analysis of variance. Results The level of TSLP protein was （155.5 ± 5.9） ng/L, （228.8 ± 28.7） ng/L, and （166.2 ± 8.8） ng/L in the culture supernatant of keratinocytes after 12-hour treatment with rDer p1 of 1 mg/L and 10 mg/L as well as SLGIKV respectively, significantly higher than that in the blank control group （（54.3 ± 13.9） ng/L, all P 〈 0.01）. The TSLP mRNA expression in keratinocytes peaked at 8 hours after the stimulation with rDer p1 of 10 mg/L and SLGIKV separately, with the relative expression levels being （3.28 ± 0.27） folds and （2.15 ± 0.26） folds, respectively, which were significantly different from those at 4 and 24 hours （all P 〈 0.01）. Both SLGIKV and rDer p1 of 10 mg/L promoted intracellular calcium influx in keratinocytes, while the pretreatment with VKGILS obviously inhibited the rDer p1-induced increase in intracellular calcium influx. Conclusions The house dust mite allergen rDer p1 may induce the production of the pro-inflammatory cytokine TSLP by human keratinocytes partly via activating PAR2 receptors.