胱天蛋白酶募集域蛋白9缺乏对小鼠烟曲霉角膜炎的影响  

Effect of caspase recruitment domain-containing protein 9 deficiency on aspergillus fumigatus keratitis in mice

作　　者：吴赛男 齐晓暄 杨雅淳 熊丹瑜 林步云 张青[1] Wu Sainan;Qi Xiaoxuan;Yang Yachun;Xiong Danyu;Lin Buyun;Zhang Qing(Dept of Ophthalmology,The Second Affiliated Hospital of Anhui Medical University,Hefei 230601)

机构地区：[1]安徽医科大学第二附属医院眼科,合肥230601

出　　处：《安徽医科大学学报》2025年第3期446-454,共9页Acta Universitatis Medicinalis Anhui

基　　金：安徽省高校自然科学研究项目(编号:KJ2020A0180);安徽省教育厅省级研究生学术创新项目(编号:2022xscx059);安徽省教育厅省级研究生教育教学改革研究项目(编号:2022jyjxggyj226);安徽省卫生健康科研项目(编号:AHWJ2023A20229);安徽医科大学第二附属医院首批国家自然科学基金孵育计划项目(编号:2019GQFY04);安徽医科大学第二附属医院第四批科技新星培优项目(编号:2018KA08);2020年高校优秀青年骨干人才国外访学研修项目(编号:gxgwfx2020024)。

摘　　要：目的研究胱天蛋白酶募集域蛋白9(Card9)在烟曲霉性角膜炎中的作用,以及Card9缺乏对巨噬细胞抵御真菌感染的影响。方法(1)选择6~8周的C57BL/7小鼠,分别用Card9 siRNA和Blank siRNA进行预处理,通过Western blot和RT-PCR检测各组Card9的表达;siRNA预处理72 h后刮去角膜上皮,注射烟曲霉孢子悬液,在感染后第1、3、5、7天记录角膜评分,通过RT-PCR、免疫组化检测法(IHC)检测各组Card9、核因子κB(NF-κB)、白细胞介素1β(IL-1β)、IL-6、肿瘤坏死因子α(TNF-α)的表达;(2)体外培养人角膜上皮细胞(HCECs)、人单核巨噬细胞(THP-1细胞),使用RT-PCR检测两种细胞的Card9基因的表达;使用shRNA载体对THP-1细胞构建稳转细胞系,通过Western blot和RT-PCR检测Card9的表达,诱导成巨噬细胞,烟曲霉刺激后用RT-PCR检测Card9、NF-κB、IL-1β、IL-6、TNF-α的表达。结果小鼠烟曲霉性角膜炎中Card9表达增加,主要分布在免疫细胞的细胞质中;使用Card9 siRNA处理后的小鼠角膜中Card9表达明显减少;在抑制Card9基因的表达后,IL-1β、IL-6、TNF-α、NF-κB的表达明显减少,其中以IL-1β的变化最为显著。体外实验中,HCECs中几乎不表达Card9,而THP-1细胞中大量表达;诱导后的巨噬细胞在烟曲霉刺激下,Card9、NF-κB、IL-1β、IL-6、TNF-α均显著上调,抑制Card9表达后,刺激后的表达明显减少,以IL-1β的变化最为显著。结论Card9参与烟曲霉性角膜炎的炎症发展及愈合过程,Card9缺乏可引起巨噬细胞功能受损,并抑制炎症因子表达,其中IL-1β的影响最大。Objective To investigate the role of caspase recruitment domain-containing protein 9(Card9)in Aspergillus fumigatus(A.fumigatus)keratitis and the effect of its deficiency on macrophage resistance to fungal infection.Methods①C57BL/7 mice aged 6-8 weeks were selected and the mice pretreated Card9 siRNA and Blank siRNA,respectively,and the expression of Card9 in each group was detected by Western blot and RT-PCR.The corneal epithelium of the mice was scraped away 72 hours later,and A.fumigatus spore suspension was injected into the corneal stroma.The corneal scores were recorded at 1 d,3 d,5 d and 7 d after infection.The expression of Card9,nuclear factorκB(NF-κB),interleukin 1β(IL-1β),interleukin 6(IL-6)and tumor necrosis factorα(TNF-α)in each group was detected by RT-PCR and immunohistochemical(IHC).②Human corneal epithelial cells(HCECs)and human monocytic-leukemia cells(THP-1)in vitro,RT-PCR was used to examine the expression of Card9 gene in the two cells,and a stable cell line of THP-1 cells was constructed using shRNA vectors.The expression of Card9 in the cell line was detected by Western Blot and RT-PCR.The cells were induced into macrophages and stimulation by A.fumigatus,and the expression of Card9,NF-κB,IL-1β,IL-6 and TNF-αwas detected by RT-PCR.Results Card9 expression increased in A.fumigatus keratitis,mainly distributed in cytoplasm of immune cells.The expression of Card9 in the cornea of mice treated with Card9 siRNA was significantly reduced.After inhibiting the expression of Card9 gene,the expressions of Card9,NF-κB,IL-1β,IL-6 and TNF-αsignificantly decreased and the changes of IL-1βwere most significant.In in vitro studies,Card9 exhibited negligible expression in human corneal epithelial cells,contrasting with its pronounced expression in THP-1 cells.After the induction of macrophages,Card9,NF-κB,IL-1β,IL-6,and TNF-αwere significantly upregulated under the stimulation of A.fumigatus.After inhibiting the expression of Card9,the stimulated expression of these factors was significa

关 键 词：Card9 烟曲霉菌 巨噬细胞 炎症因子 角膜炎 

分 类 号：R772.21[医药卫生—眼科]