LOX-1在小鼠真菌性角膜炎中對粘附因子調控的實驗研究
發(fā)布時間：2019-01-02 11:20
【摘要】：目的:通過觀察凝集素樣氧化性低密度脂蛋白受體-1(lectin-like oxidized low density lipoprotein recepter-1,LOX-1)在小鼠真菌性角膜炎過程中對粘附因子的調控作用,探討LOX-1對小鼠角膜中性粒細胞遷移和募集的影響。方法:煙曲霉菌刺激后建立SPF級C57BL/6小鼠真菌性角膜炎模型,分別于刺激后12小時、1天、3天裂隙燈顯微鏡下觀察小鼠角膜炎癥反應,收集全角膜;采用硫代乙醇酸鹽溶液刺激法從小鼠腹腔中提取中性粒細胞,煙曲霉菌刺激細胞4、8、12小時后觀察細胞情況并收集細胞;分別用LOX-1中和抗體、抑制劑預處理小鼠角膜和小鼠腹腔中性粒細胞,以Ig G、PBS為對照,同樣方法建立小鼠真菌性角膜炎模型和中性粒細胞刺激模型。分別采用臨床炎癥評分評價各組小鼠角膜感染程度;采用real-time RT-PCR技術檢測各組小鼠角膜中ICAM-1、VCAM-1、P-selectin、E-selectin、IL-8 m RNA的表達變化;采用免疫熒光半定量和定位小鼠角膜中ICAM-1蛋白的表達變化和部位;采用western bloting定量檢測各組小鼠角膜中ICAM-1及中性粒細胞中LFA-1的蛋白表達變化;采用免疫熒光及MPO檢測各組小鼠角膜中中性粒細胞的數(shù)量變化。結果:LOX-1中和抗體預處理組和LOX-1抑制劑預處理組小鼠角膜炎癥反應減輕,臨床炎癥評分較Ig G組和PBS組降低;煙曲霉菌感染小鼠角膜12h、1d、3d后,ICAM-1、VCAM-1、P-selectin、E-selectin和IL-8基因和蛋白的表達較正常小鼠角膜升高;LOX-1中和抗體預處理組和LOX-1抑制劑預處理組煙曲霉菌感染小鼠角膜12h、1d、3d后,ICAM-1、VCAM-1、P-selectin、E-selectin和IL-8基因和蛋白的表達較Ig G組和PBS組降低;LOX-1中和抗體和LOX-1抑制劑預處理小鼠腹腔中性粒細胞2h,再給予煙曲霉菌刺激4h、8h、12h后,LFA-1基因和蛋白的表達較Ig G組和PBS組降低;經LOX-1中和抗體和LOX-1抑制劑預處理1d,再給予煙曲霉菌感染小鼠角膜3d后,小鼠角膜中中性粒細胞數(shù)量及MPO相對表達量較Ig G組和PBS組減少。結論:LOX-1可調控小鼠煙曲霉菌性角膜炎中粘附因子的表達,通過影響小鼠角膜中性粒細胞的遷移和募集參與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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R772.21
本文編號：2398415
[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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R772.21
【參考文獻】
相關期刊論文 前3條
1 Kun He;Li-Hui Yue;Gui-Qiu Zhao;Cui Li;Jing Lin;Nan Jiang;Qian Wang;Qiang Xu;Xu-Dong Peng;Li-Ting Hu;Jie Zhang;;The role of LOX-1 on innate immunity against Aspergillus keratitis in mice[J];International Journal of Ophthalmology;2016年09期
2 Cui Li;Gui-Qiu Zhao;Cheng-Ye Che;Jing Lin;Na Li;Wen-Yan Jia;Qiu-QiuZhang;Nan Jiang;Li-Ting Hu;;Effect of corneal graft diameter on therapeutic penetrating keratoplasty for fungal keratitis[J];International Journal of Ophthalmology(English Edition);2012年06期
3 李娜;趙桂秋;;真菌性角膜炎免疫機制研究進展[J];中華眼科雜志;2011年04期
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