Mincle对真菌性角膜炎中性粒细胞凋亡的调控作用

发布时间：2018-11-02 07:53

【摘要】：目的:研究巨噬细胞诱导c型凝集素(Macrophage inducible C-type lectin,Mincle)在真菌性角膜炎中对中性粒细胞凋亡的调控作用。方法:随机将健康的C57BL/6小鼠分为对照组和烟曲霉菌角膜炎组,分别于建模后12小时、1天、3天、5天裂隙灯显微镜下观察小鼠角膜感染情况,收集全角膜,检测Mincle的表达情况。用Mincle激动剂TDB以及Mincle中和抗体球结膜下注射预处理后,然后在烟曲霉菌感染小鼠角膜1天及3天,观察小鼠角膜感染情况,收集全角膜,q RT-PCR法检测小鼠角膜中Mincle、FAS、FASL和Caspase3 m RNA的表达,免疫荧光检测小鼠角膜中性粒细胞的浸润和细胞凋亡情况。从小鼠腹腔中提取中性粒细胞,并用烟曲霉菌刺激4小时、8小时、12小时、16小时,q RT-PCR法检测中性粒细胞中Mincle m RNA的表达。TDB和Mincle的中和抗体预处理小鼠腹腔中性粒细胞2小时,然后用灭活的烟曲霉菌刺激中性粒细胞8小时,q RT-PCR法检测FAS、FASL、Caspase3 m RNA的表达,Western blot检测Caspase3的蛋白表达,流式细胞仪检测炎症因子和中性粒细胞的凋亡情况。结果:灭活的烟曲霉菌感染C57BL/6小鼠12小时、1天、3天、5天后角膜中Mincle的m RNA和蛋白比正常对照组中的表达高,在12小时Mincle的m RNA表达最高。TDB处理组和DMSO处理组相比,TDB组Mincle的m RNA表达增多。TDB预处理组烟曲霉菌感染小鼠角膜1天后,Fas、Fasl和Caspase3的m RNA水平与单纯的烟曲霉菌刺激相比显著降低;Ig G预处理组烟曲霉菌感染1天后Fas、Fasl和Caspase3的m RNA水平与Mincle中和抗体预处理组相比显著降低。TDB处理组和DMSO处理组相比,Mincle的蛋白表达升高,TUNEL染色阳性细胞数减少,中性粒细胞数目增多。TDB预处理组烟曲霉菌感染小鼠角膜3天与单纯的烟曲霉菌刺激相比,Fas、Fasl和Caspase3的蛋白水平显著降低,TUNEL染色阳性细胞数减少,而中性粒细胞数目是增多的;Ig G预处理组烟曲霉菌感染3天与Mincle中和抗体预处理组烟曲霉菌感染3天相比Fas、Fasl和Caspase3的蛋白水平显著降低,TUNEL染色阳性细胞数减少,而中性粒细胞数目是增多的。灭活的烟曲霉菌刺激中性粒细胞4小时、8小时、12小时、16小时后Mincle的m RNA和蛋白比正常对照组中的表达高,在8小时Mincle的m RNA表达水平最高。TDB处理组和DMSO处理组相比,TDB组Mincle的m RNA表达增多。TDB预处理组烟曲霉菌刺激小鼠中性粒细胞8小时后,Fas、Fasl和Caspase3的m RNA和蛋白表达水平与单纯的烟曲霉菌刺激相比显著降低,中性粒细胞凋亡率下降;Ig G预处理组烟曲霉菌刺激8小时后Fas、Fasl和Caspase3的m RNA和蛋白表达水平与Mincle中和抗体预处理组相比显著降低,中性粒细胞的凋亡率下降。结论:Mincle参与烟曲霉菌性角膜炎的炎症反应过程,真菌感染后Mincle的表达上调同时中性粒细胞的浸润水平和凋亡数目也是增加的。Mincle还可以通过抑制FAS和FASL的结合来抑制Caspase3的表达进而抑制嗜中性粒细胞凋亡。
[Abstract]:Aim: to investigate the role of macrophage-induced c-lectin (Macrophage inducible C-type lectin,Mincle in the regulation of neutrophil apoptosis in fungal keratitis. Methods: healthy C57BL/6 mice were randomly divided into control group and aspergillus fumigatus group. The corneal infection was observed under slit lamp microscope at 12 hours, 1 day, 3 days and 5 days after modeling, and the whole cornea was collected. The expression of Mincle was detected. After pre-treatment with Mincle agonist TDB and Mincle neutralizing antibody subconjunctival injection, the cornea of mice infected with Aspergillus fumigatus were observed for 1 and 3 days, and the whole cornea was collected. Q RT-PCR method was used to detect Mincle,FAS, in the cornea of mice. The expression of FASL and Caspase3 m RNA, the infiltration of neutrophil and apoptosis of mouse cornea were detected by immunofluorescence. Neutrophils were extracted from the abdominal cavity of mice and stimulated with Aspergillus fumigatus for 4 hours, 8 hours, 12 hours, 16 hours, respectively. Q RT-PCR assay was used to detect the expression of Mincle m RNA in neutrophils. Neutrophils were pretreated with neutralizing antibodies of TDB and Mincle for 2 hours, then stimulated with inactivated aspergillus fumigatus for 8 hours. FAS,FASL, was detected by Q RT-PCR method. The expression of Caspase3 m RNA was detected by, Western blot, the expression of Caspase3 protein was detected by, Western blot, and the apoptosis of inflammatory factors and neutrophils was detected by flow cytometry. Results: the expression of m RNA and protein of Mincle in the cornea of C57BL/6 mice infected with inactivated Aspergillus fumigatus for 12 hours, 1 day, 3 days and 5 days was higher than that in normal control group, and the expression of m RNA was highest in 12 hour Mincle group. The expression of m RNA in TDB treatment group was higher than that in DMSO treatment group. The expression of m RNA in Mincle of TDB group was increased, and the m RNA levels of Fas,Fasl and Caspase3 in TDB pretreatment group were significantly lower than those in single Aspergillus fumigatus stimulation group after 1 day of TDB preconditioning in mice cornea infected with Aspergillus fumigatus. The m RNA levels of Fas,Fasl and Caspase3 in Ig G pretreatment group were significantly lower than those in Mincle neutralizing antibody preconditioning group 1 day after infection. The expression of Mincle protein increased and the number of TUNEL staining positive cells decreased in TDB treatment group and DMSO treatment group. Compared with those stimulated by Aspergillus fumigatus alone for 3 days, the protein levels of Fas,Fasl and Caspase3 were significantly decreased, and the number of TUNEL staining positive cells was decreased in TDB pretreatment group. The number of neutrophils was increased. The protein levels of Fas,Fasl and Caspase3 in the Ig G pretreatment group were significantly lower than those in the Mincle neutralizing antibody pretreatment group on the 3rd day, and the number of TUNEL staining positive cells was decreased, while the number of neutrophils was increased. Inactivated Aspergillus fumigatus stimulated neutrophils for 4 hours, 8 hours, 12 hours, 16 hours later, the expression of m RNA and protein in Mincle was higher than that in normal control group, and the expression level of m RNA in 8 hours Mincle was the highest. The expression level of m RNA in TDB treatment group was higher than that in DMSO treatment group. The expression of m RNA in Mincle in TDB group was increased, and the expression of m RNA and protein in Fas,Fasl and Caspase3 were significantly decreased and the apoptosis rate of neutrophils was decreased in TDB pretreatment group compared with those stimulated by Aspergillus fumigatus alone. The expression of m RNA and protein of Fas,Fasl and Caspase3 in Ig G pretreatment group was significantly lower than that in Mincle neutralizing antibody pretreatment group, and the apoptosis rate of neutrophils was decreased after 8 hours of stimulation by Aspergillus fumigatus. Conclusion: Mincle is involved in the inflammatory reaction of tobacco curly fungal keratitis. The expression of Mincle was up-regulated after fungal infection, and the infiltration and apoptosis of neutrophils were also increased. Mincle could also inhibit the expression of Caspase3 and then inhibit the apoptosis of neutrophils by inhibiting the binding of FAS and FASL.
【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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