Che Cheng-Ye, Jia Wen-Yan, Xu Qiang, Li Na, Hu Li-Ting, Jiang Nan, Lin Jing, Wang Qing, Zhao Gui-Qiu
Department of Ophthalmology, the Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China.
Int J Ophthalmol. 2012;5(1):13-7. doi: 10.3980/j.issn.2222-3959.2012.01.03. Epub 2012 Feb 18.
To investigate roles of surfactant protein D (SP-D) and relative cytokines in human corneal epithelial(HCE) cells exposed to aspergillus fumigatus (AF) antigens.
HCE cells cultured in vitro with AF antigens and sampled at 0, 0.5, 1 hour, 2, 4, 6 and 8 hours. The expression of SP-D mRNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).The expression of SP-D protein was shown by ELISA and immunocytochemistry SP methods. The expression of NF-κB and relative downstream cytokines such as TNF-α, IL-1β, IL-8 and IL-10 in supernatant fluid were measured by ELISA.
SP-D mRNA and protein were detected in untreated HCE cells. The expression of SP-D and the relative downstream cytokines rose after being stimulated with AF antigens. SP-D mRNA began to rise at 0.5 hour and the most significantly peak was in 2 hours. The protein of SP-D in supernatant fluid had the same trend with mRNA. Immunocytochemistry of SP-D showed positive expression and gradually increased to 6 hours, and then the expression began to decline. NF-κB was activated after treated by AF antigens and the changes had correlation with SP-D. TNF-α, IL-1β, IL-8 and IL-10 began to rise after given AF antigens 1 hour and were 1.82, 1.43, 1.12 and 1.28 times higher than the untreated HCE cells separately. The expression of TNF-α and IL-1β reached the peak at 2 hours, separately 2.80 and 2.86 times than the untreated. The expression of IL-8 and IL-10 gradually increased with a time-dependent manner.
HCE cells exists SP-D and it may play a significant role in pathogenesis of keratomycosis. AF may induce human corneal epithelial cells to express inflammatory cytokines via SP-D and NF-κB pathway. SP-D possibly mediates the recognition to AF mycelium.
研究表面活性蛋白D（SP-D）及相关细胞因子在暴露于烟曲霉（AF）抗原的人角膜上皮（HCE）细胞中的作用。
体外培养HCE细胞，用AF抗原处理，并在0、0.5、1小时、2、4、6和8小时取样。通过半定量逆转录-聚合酶链反应（RT-PCR）评估SP-D mRNA的表达。用ELISA和免疫细胞化学SP法显示SP-D蛋白的表达。用ELISA测定上清液中NF-κB及相关下游细胞因子如TNF-α、IL-1β、IL-8和IL-10的表达。
在未处理的HCE细胞中检测到SP-D mRNA和蛋白。AF抗原刺激后，SP-D及相关下游细胞因子的表达升高。SP-D mRNA在0.5小时开始升高，最显著峰值出现在2小时。上清液中SP-D蛋白与mRNA趋势相同。SP-D免疫细胞化学显示阳性表达，并逐渐增加至6小时，然后表达开始下降。AF抗原处理后NF-κB被激活，其变化与SP-D相关。给予AF抗原1小时后，TNF-α、IL-1β、IL-8和IL-10开始升高，分别比未处理的HCE细胞高1.82、1.43、1.12和1.28倍。TNF-α和IL-1β的表达在2小时达到峰值，分别是未处理的2.80和2.86倍。IL-8和IL-10的表达随时间呈逐渐增加的趋势。
HCE细胞存在SP-D，其可能在角膜真菌病发病机制中起重要作用。AF可能通过SP-D和NF-κB途径诱导人角膜上皮细胞表达炎性细胞因子。SP-D可能介导对AF菌丝体的识别。
