【摘要】：目的:探究環(huán)孢素A(Cyclosporine A,Cs A)在煙曲霉菌(Aspergillus Fumigatus,A.fumigatus)感染的人角膜上皮細(xì)胞中對Dectin-1表達(dá)的影響,并進(jìn)一步探究維生素D(1,25(OH)2D3)及維生素D受體(Vitamin D Receptor,VDR)信號通路對環(huán)孢素A作用的影響。方法:Cs A預(yù)處理永生化人角膜上皮細(xì)胞(Immortalized human corneal epithelial cells,HCECs)12小時,煙曲霉菌或凝膠多糖(Curdlan)分別刺激HCECs后,應(yīng)用熒光定量PCR(rt PCR)技術(shù)檢測Dectin-1受體m RNA的表達(dá),并應(yīng)用Western Blot技術(shù)檢測Dectin-1受體蛋白的表達(dá);分別應(yīng)用外源性1,25(OH)2D3及VDR抗體預(yù)處理HCECs1小時,再應(yīng)用Cs A處理12小時,最后應(yīng)用煙曲霉菌及凝膠多糖分別刺激HCECs后,應(yīng)用rt PCR及Western Blot技術(shù)檢測Dectin-1的表達(dá),同時應(yīng)用酶聯(lián)免疫吸附試驗(ELISA)檢測不同刺激條件下炎癥因子IL-1β及TNF-α的表達(dá)變化。結(jié)果:煙曲霉菌及凝膠多糖分別刺激HCECs后,Dectin-1受體m RNA及蛋白表達(dá)均顯著升高,Cs A預(yù)處理后能顯著抑制Dectin-1表達(dá)的升高。VDR抑制劑及Cs A預(yù)處理HCECs后,Cs A對Dectin-1表達(dá)的抑制作用與單純應(yīng)用Cs A刺激相比明顯減弱,1,25(OH)2D3及Cs A預(yù)處理HCECs后,Cs A對Dectin-1表達(dá)的抑制作用與單純應(yīng)用Cs A刺激相比明顯增強(qiáng),炎癥因子也呈現(xiàn)出相同的變化。結(jié)論:煙曲霉菌或凝膠多糖刺激后,Cs A能夠抑制Dectin-1及下游炎癥因子的表達(dá),維生素D的活性形式1,25(OH)2D3及VDR信號通路能夠調(diào)節(jié)Cs A的抑制作用。1,25(OH)2D3增強(qiáng)此種抑制作用而VDR抑制劑減弱此種抑制作用。
[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- 偽 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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R772.21

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