环孢素A在角膜上皮细胞抗真菌免疫中对Dectin-1表达的影响及机制
发布时间：2018-08-25 16:38
【摘要】：目的:探究环孢素A(Cyclosporine A,Cs A)在烟曲霉菌(Aspergillus Fumigatus,A.fumigatus)感染的人角膜上皮细胞中对Dectin-1表达的影响,并进一步探究维生素D(1,25(OH)2D3)及维生素D受体(Vitamin D Receptor,VDR)信号通路对环孢素A作用的影响。方法:Cs A预处理永生化人角膜上皮细胞(Immortalized human corneal epithelial cells,HCECs)12小时,烟曲霉菌或凝胶多糖(Curdlan)分别刺激HCECs后,应用荧光定量PCR(rt PCR)技术检测Dectin-1受体m RNA的表达,并应用Western Blot技术检测Dectin-1受体蛋白的表达;分别应用外源性1,25(OH)2D3及VDR抗体预处理HCECs1小时,再应用Cs A处理12小时,最后应用烟曲霉菌及凝胶多糖分别刺激HCECs后,应用rt PCR及Western Blot技术检测Dectin-1的表达,同时应用酶联免疫吸附试验(ELISA)检测不同刺激条件下炎症因子IL-1β及TNF-α的表达变化。结果:烟曲霉菌及凝胶多糖分别刺激HCECs后,Dectin-1受体m RNA及蛋白表达均显著升高,Cs A预处理后能显著抑制Dectin-1表达的升高。VDR抑制剂及Cs A预处理HCECs后,Cs A对Dectin-1表达的抑制作用与单纯应用Cs A刺激相比明显减弱,1,25(OH)2D3及Cs A预处理HCECs后,Cs A对Dectin-1表达的抑制作用与单纯应用Cs A刺激相比明显增强,炎症因子也呈现出相同的变化。结论:烟曲霉菌或凝胶多糖刺激后,Cs A能够抑制Dectin-1及下游炎症因子的表达,维生素D的活性形式1,25(OH)2D3及VDR信号通路能够调节Cs A的抑制作用。1,25(OH)2D3增强此种抑制作用而VDR抑制剂减弱此种抑制作用。
[Abstract]:Aim: to investigate the effect of cyclosporine (A (Cyclosporine) on the expression of Dectin-1 in human corneal epithelial cells infected with Aspergillus fumigatus (Aspergillus Fumigatus,A.fumigatus), and to explore the effects of vitamin D (1: 25 (OH) 2D3) and vitamin D receptor (Vitamin D Receptor,VDR) signaling pathway on cyclosporine A (CSA) expression in human corneal epithelial cells infected with Aspergillus fumigatus (Aspergillus Fumigatus,A.fumigatus). Methods (Immortalized human corneal epithelial cells,HCECs of immortalized human corneal epithelial cells was pretreated with 10% CSA for 12 hours. After HCECs was stimulated by Aspergillus fumigatus or (Curdlan), the expression of Dectin-1 receptor m RNA was detected by fluorescence quantitative PCR (rt PCR) technique. Western Blot technique was used to detect the expression of Dectin-1 receptor protein, the HCECs1 was pretreated with exogenous 1 25 (OH) 2D3 and VDR antibody for 12 hours, then treated with Cs A for 12 hours. Finally, HCECs was stimulated by Aspergillus fumigatus and gelatin polysaccharides, respectively. The expression of Dectin-1 was detected by rt PCR and Western Blot, and the expression of IL-1 尾 and TNF- 伪 was detected by Elisa (ELISA). Results: after stimulation of HCECs by Aspergillus fumigatus and gel polysaccharides, the expression of m RNA and protein of Dectin-1 receptor increased significantly. VDR inhibitor and Cs A pretreatment with HCECs inhibited Dectin-1 expression significantly. Compared with the stimulation of Cs A alone, the inhibitory effect of CSA on the expression of Dectin-1 was significantly decreased after pretreatment of HCECs with Cs A, and the inhibitory effect of CSA on Dectin-1 expression was significantly increased compared with the stimulation of Cs A alone. The same changes were observed in inflammatory factors. Conclusion: after stimulation by Aspergillus fumigatus or gel polysaccharide, CSA can inhibit the expression of Dectin-1 and its downstream inflammatory factors. The active form of vitamin D (1: 25 (OH) 2D3 and VDR signaling pathway) could modulate the inhibitory effect of Cs A. the inhibitory effect of Cs A was enhanced by 1 ~ (25) (OH) 2D3, while the inhibitory effect of VDR inhibitor was weakened.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R772.21
本文编号：2203488
[Abstract]:Aim: to investigate the effect of cyclosporine (A (Cyclosporine) on the expression of Dectin-1 in human corneal epithelial cells infected with Aspergillus fumigatus (Aspergillus Fumigatus,A.fumigatus), and to explore the effects of vitamin D (1: 25 (OH) 2D3) and vitamin D receptor (Vitamin D Receptor,VDR) signaling pathway on cyclosporine A (CSA) expression in human corneal epithelial cells infected with Aspergillus fumigatus (Aspergillus Fumigatus,A.fumigatus). Methods (Immortalized human corneal epithelial cells,HCECs of immortalized human corneal epithelial cells was pretreated with 10% CSA for 12 hours. After HCECs was stimulated by Aspergillus fumigatus or (Curdlan), the expression of Dectin-1 receptor m RNA was detected by fluorescence quantitative PCR (rt PCR) technique. Western Blot technique was used to detect the expression of Dectin-1 receptor protein, the HCECs1 was pretreated with exogenous 1 25 (OH) 2D3 and VDR antibody for 12 hours, then treated with Cs A for 12 hours. Finally, HCECs was stimulated by Aspergillus fumigatus and gelatin polysaccharides, respectively. The expression of Dectin-1 was detected by rt PCR and Western Blot, and the expression of IL-1 尾 and TNF- 伪 was detected by Elisa (ELISA). Results: after stimulation of HCECs by Aspergillus fumigatus and gel polysaccharides, the expression of m RNA and protein of Dectin-1 receptor increased significantly. VDR inhibitor and Cs A pretreatment with HCECs inhibited Dectin-1 expression significantly. Compared with the stimulation of Cs A alone, the inhibitory effect of CSA on the expression of Dectin-1 was significantly decreased after pretreatment of HCECs with Cs A, and the inhibitory effect of CSA on Dectin-1 expression was significantly increased compared with the stimulation of Cs A alone. The same changes were observed in inflammatory factors. Conclusion: after stimulation by Aspergillus fumigatus or gel polysaccharide, CSA can inhibit the expression of Dectin-1 and its downstream inflammatory factors. The active form of vitamin D (1: 25 (OH) 2D3 and VDR signaling pathway) could modulate the inhibitory effect of Cs A. the inhibitory effect of Cs A was enhanced by 1 ~ (25) (OH) 2D3, while the inhibitory effect of VDR inhibitor was weakened.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R772.21
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