LOX-1在小鼠真菌性角膜炎中对粘附因子调控的实验研究

发布时间：2019-01-02 11:20

【摘要】：目的:通过观察凝集素样氧化性低密度脂蛋白受体-1(lectin-like oxidized low density lipoprotein recepter-1,LOX-1)在小鼠真菌性角膜炎过程中对粘附因子的调控作用,探讨LOX-1对小鼠角膜中性粒细胞迁移和募集的影响。方法:烟曲霉菌刺激后建立SPF级C57BL/6小鼠真菌性角膜炎模型,分别于刺激后12小时、1天、3天裂隙灯显微镜下观察小鼠角膜炎症反应,收集全角膜;采用硫代乙醇酸盐溶液刺激法从小鼠腹腔中提取中性粒细胞,烟曲霉菌刺激细胞4、8、12小时后观察细胞情况并收集细胞;分别用LOX-1中和抗体、抑制剂预处理小鼠角膜和小鼠腹腔中性粒细胞,以Ig G、PBS为对照,同样方法建立小鼠真菌性角膜炎模型和中性粒细胞刺激模型。分别采用临床炎症评分评价各组小鼠角膜感染程度;采用real-time RT-PCR技术检测各组小鼠角膜中ICAM-1、VCAM-1、P-selectin、E-selectin、IL-8 m RNA的表达变化;采用免疫荧光半定量和定位小鼠角膜中ICAM-1蛋白的表达变化和部位;采用western bloting定量检测各组小鼠角膜中ICAM-1及中性粒细胞中LFA-1的蛋白表达变化;采用免疫荧光及MPO检测各组小鼠角膜中中性粒细胞的数量变化。结果:LOX-1中和抗体预处理组和LOX-1抑制剂预处理组小鼠角膜炎症反应减轻,临床炎症评分较Ig G组和PBS组降低;烟曲霉菌感染小鼠角膜12h、1d、3d后,ICAM-1、VCAM-1、P-selectin、E-selectin和IL-8基因和蛋白的表达较正常小鼠角膜升高;LOX-1中和抗体预处理组和LOX-1抑制剂预处理组烟曲霉菌感染小鼠角膜12h、1d、3d后,ICAM-1、VCAM-1、P-selectin、E-selectin和IL-8基因和蛋白的表达较Ig G组和PBS组降低;LOX-1中和抗体和LOX-1抑制剂预处理小鼠腹腔中性粒细胞2h,再给予烟曲霉菌刺激4h、8h、12h后,LFA-1基因和蛋白的表达较Ig G组和PBS组降低;经LOX-1中和抗体和LOX-1抑制剂预处理1d,再给予烟曲霉菌感染小鼠角膜3d后,小鼠角膜中中性粒细胞数量及MPO相对表达量较Ig G组和PBS组减少。结论:LOX-1可调控小鼠烟曲霉菌性角膜炎中粘附因子的表达,通过影响小鼠角膜中性粒细胞的迁移和募集参与小鼠角膜抗真菌固有免疫。
[Abstract]:Aim: to investigate the role of lectin-like oxidized low density lipoprotein receptor-1 (lectin-like oxidized low density lipoprotein recepter-1,LOX-1) in the regulation of adhesion factor during fungal keratitis in mice. To investigate the effect of LOX-1 on neutrophil migration and recruitment in mice. Methods: the fungal keratitis model of SPF grade C57BL/6 mice was established after stimulation by Aspergillus fumigatus. The inflammatory reaction of mice cornea was observed under slit lamp microscope at 12 hours, 1 day and 3 days after stimulation, and the whole cornea was collected. Neutrophils were extracted from mouse abdominal cavity by thioglycolate solution stimulation method. After stimulation by Aspergillus fumigatus, the cells were observed and collected 12 hours after stimulation by Aspergillus fumigatus. LOX-1 neutralizing antibody and inhibitor were used to pretreat mouse cornea and mouse peritoneal neutrophils, respectively. The mice fungal keratitis model and neutrophil stimulation model were established by using Ig GnP PBS as control. The clinical inflammation score was used to evaluate the degree of corneal infection in each group, and the expression of ICAM-1,VCAM-1,P-selectin,E-selectin,IL-8 m RNA in the cornea of each group was detected by real-time RT-PCR technique. The expression and location of ICAM-1 protein in the cornea of mice were detected by immunofluorescence semi-quantification and localization, and the expression of LFA-1 protein in cornea and neutrophils in each group was quantitatively detected by western bloting. The number of corneal neutrophils in each group was detected by immunofluorescence and MPO. Results: the corneal inflammation in LOX-1 neutralizing antibody preconditioning group and LOX-1 inhibitor pretreatment group was reduced, and the clinical inflammatory score was lower than that in Ig G group and PBS group. The expression of ICAM-1,VCAM-1,P-selectin,E-selectin and IL-8 gene and protein in the cornea of mice infected with Aspergillus fumigatus for 12 h or 1 d were higher than those in normal cornea. The expression of ICAM-1,VCAM-1,P-selectin,E-selectin and IL-8 gene and protein in LOX-1 neutralizing antibody preconditioning group and LOX-1 inhibitor pretreatment group were lower than those in Ig G group and PBS group. The expression of LFA-1 gene and protein in LOX-1 neutralizing antibody and LOX-1 inhibitor was lower than that in Ig G group and PBS group after pretreatment for 2 h and then stimulated by Aspergillus fumigatus for 4 h or 8 h for 12 h. After pretreatment with LOX-1 neutralizing antibody and LOX-1 inhibitor for 1 day, the number of neutrophils and the relative expression of MPO in the cornea of mice infected with Aspergillus fumigatus for 3 days were lower than those in Ig G group and PBS group. Conclusion: LOX-1 can regulate the expression of adhesion factor in mice with tobacco-mycetes keratitis and participate in the innate immunity of mouse cornea by affecting the migration and recruitment of corneal neutrophils.
【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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