真菌性角膜炎角膜组织NOD1、NF-κBp65表达的变化以及炎症细胞浸润情况  

Changes in expression of nucleotide-binding oligomerization domain-containing protein 1 and nuclear factor kappa-Bp65 and infiltration of inflammatory cells in the cornea with fungal keratitis

作　　者：李妍[1] 李丽红[2,3] 戴敏 闵婕[1] 胡竹林 LI Yan;LI Lihong;DAI Min;MIN Jie;HU Zhulin(The Yunnan University Affiliated Hospital(the Second People’s Hospital of Yunnan Province,Eye Hospital of Yunnan Province),Yunnan Eye Institute,the Key Laboratory of Yunnan Province for Ophthalmic Research and Disease Control,Kunming 650021,Yunnan Province,China;The Laboratory Animal Centre,Kunming Institute of Zoology,Chinese Academy of Sciences,Kunming 650201,Yunnan Province,China;The National Resource Center for Non-Human Primates,Kunming 650199,Yunnan Province,China)

机构地区：[1]云南大学附属医院(云南省第二人民医院,云南省眼科医院)眼科,云南省眼科研究所,云南省眼科疾病防治研究重点实验室,云南省昆明市650021 [2]中国科学院昆明动物研究所实验动物中心,云南省昆明市650201 [3]国家非人灵长类实验动物资源库,云南省昆明市650199

出　　处：《眼科新进展》2023年第2期89-93,共5页Recent Advances in Ophthalmology

基　　金：云南省基础研究专项(编号:202201AT070073);云南省科技厅昆医联合专项[编号:202001AY070001(-165)]。

摘　　要：目的探讨真菌性角膜炎角膜组织NOD1、NF-κBp65的表达变化以及炎症细胞浸润情况。方法取清洁级树鼩25只分成3组,实验组10只,空白对照组10只,正常对照组5只,均取右眼为实验眼。将茄病镰刀菌接种到沙氏葡萄糖琼脂培养基中培养,收集真菌孢子混悬液。实验组用胰岛素针头(29G)在实验眼角膜上皮细胞层做“#”形划痕,结膜囊滴真菌孢子混悬液5μL;空白对照组实验眼结膜囊滴生理盐水5μL,其余步骤同实验组;正常对照组不做任何处理。采用Western blot检测蛋白表达,HE染色观察炎症细胞浸润情况,免疫组织化学染色检测蛋白阳性表达情况。结果Western blot检测结果显示:实验组角膜组织中NOD1及NF-κBp65的蛋白相对表达量均明显升高,与正常对照组及空白对照组相比,差异均有统计学意义(均为P<0.05);正常对照组角膜组织中NOD1及NF-κBp65的蛋白相对表达量与空白对照组相比,差异均无统计学意义(均为P>0.05)。免疫组织化学染色结果显示:正常对照组及空白对照组角膜上皮细胞胞浆均未见NOD1蛋白表达,实验组NOD1蛋白表达于角膜上皮细胞胞浆,表达强度与时间呈正相关;正常对照组及空白对照组角膜基质层及角膜内皮细胞层均未见NF-κBp65蛋白表达,实验组NF-κBp65蛋白表达于角膜基质层的中性粒细胞及角膜内皮细胞,表达强度与时间呈正相关。HE染色结果显示:正常对照组角膜各层组织结构正常,未见炎症细胞浸润;实验组造模后第1天,角膜水肿,上皮细胞层缺失,基质层见大量炎症细胞浸润,以中性粒细胞为主;造模后第3天,角膜高度水肿,组织形态消失,炎症细胞浸润数量明显多于第1天,同样以中性粒细胞为主。结论NOD1与NF-κBp65在树鼩镰刀菌性角膜炎角膜组织表达升高,NOD1/NF-κB信号通路在真菌性角膜炎促炎反应中发挥重要作用。Objective To discuss the expression changes of nucleotide-binding oligomerization domain-containing protein 1(NOD1)and nuclear factor kappa-Bp65(NF-κBp65)and inflammatory cell infiltration in the cornea with fungal keratitis.Methods Clean tree shrews(n=25)were randomly divided into three groups,namely the experimental group(n=10),blank control group(n=10),and normal control group(n=5).The right eye was taken as the experimental eye.The fusarium solani was inoculated into Sabouraud’s dextrose agar medium.The fungal spore suspension was collected.In the experimental group,an insulin needle(29G)was used to make a mark#on the epithelial layer of the cornea,and 5μL of fungal spore suspension was instilled in the conjunctival sac.In the blank control group,5μL of normal saline was instilled in the conjunctival sac,and other steps were the same as those of the experimental group.No treatment was made to the normal control group.The protein expression was detected through Western blot.The infiltration of inflammatory cells was observed via the hematoxylin-eosin(HE)staining and the positive expression of the protein was detected using the immunohistochemical staining.Results The result of the Western blot showed that compared with the normal control group and blank control group,the expression levels of NOD1 and NF-κBp65 were higher in the experimental group and the differences were statistically significant(all P<0.05).The relative expression levels of NOD1 and NF-κBp65 in corneal tissues of the normal control group were not significantly different from those in the blank control group(both P>0.05).Immunohistochemical staining results showed that NOD1 was not expressed in the cytoplasm of corneal epithelial cells of the normal control group and blank control group,whereas it was expressed in the cytoplasm of corneal epithelial cells of the experimental group,and the expression intensity was positively correlated with time.NF-κBp65 was not expressed in neutrophils of the corneal stroma and endothelial cells of the

关 键 词：树鼩 真菌性角膜炎 促炎反应 NOD1 NF-ΚBP65 

分 类 号：R772.2[医药卫生—眼科]