【摘要】：目的:研究巨噬細(xì)胞誘導(dǎo)c型凝集素(Macrophage inducible C-type lectin,Mincle)在真菌性角膜炎中對中性粒細(xì)胞凋亡的調(diào)控作用。方法:隨機(jī)將健康的C57BL/6小鼠分為對照組和煙曲霉菌角膜炎組,分別于建模后12小時(shí)、1天、3天、5天裂隙燈顯微鏡下觀察小鼠角膜感染情況,收集全角膜,檢測Mincle的表達(dá)情況。用Mincle激動劑TDB以及Mincle中和抗體球結(jié)膜下注射預(yù)處理后,然后在煙曲霉菌感染小鼠角膜1天及3天,觀察小鼠角膜感染情況,收集全角膜,q RT-PCR法檢測小鼠角膜中Mincle、FAS、FASL和Caspase3 m RNA的表達(dá),免疫熒光檢測小鼠角膜中性粒細(xì)胞的浸潤和細(xì)胞凋亡情況。從小鼠腹腔中提取中性粒細(xì)胞,并用煙曲霉菌刺激4小時(shí)、8小時(shí)、12小時(shí)、16小時(shí),q RT-PCR法檢測中性粒細(xì)胞中Mincle m RNA的表達(dá)。TDB和Mincle的中和抗體預(yù)處理小鼠腹腔中性粒細(xì)胞2小時(shí),然后用滅活的煙曲霉菌刺激中性粒細(xì)胞8小時(shí),q RT-PCR法檢測FAS、FASL、Caspase3 m RNA的表達(dá),Western blot檢測Caspase3的蛋白表達(dá),流式細(xì)胞儀檢測炎癥因子和中性粒細(xì)胞的凋亡情況。結(jié)果:滅活的煙曲霉菌感染C57BL/6小鼠12小時(shí)、1天、3天、5天后角膜中Mincle的m RNA和蛋白比正常對照組中的表達(dá)高,在12小時(shí)Mincle的m RNA表達(dá)最高。TDB處理組和DMSO處理組相比,TDB組Mincle的m RNA表達(dá)增多。TDB預(yù)處理組煙曲霉菌感染小鼠角膜1天后,Fas、Fasl和Caspase3的m RNA水平與單純的煙曲霉菌刺激相比顯著降低;Ig G預(yù)處理組煙曲霉菌感染1天后Fas、Fasl和Caspase3的m RNA水平與Mincle中和抗體預(yù)處理組相比顯著降低。TDB處理組和DMSO處理組相比,Mincle的蛋白表達(dá)升高,TUNEL染色陽性細(xì)胞數(shù)減少,中性粒細(xì)胞數(shù)目增多。TDB預(yù)處理組煙曲霉菌感染小鼠角膜3天與單純的煙曲霉菌刺激相比,Fas、Fasl和Caspase3的蛋白水平顯著降低,TUNEL染色陽性細(xì)胞數(shù)減少,而中性粒細(xì)胞數(shù)目是增多的;Ig G預(yù)處理組煙曲霉菌感染3天與Mincle中和抗體預(yù)處理組煙曲霉菌感染3天相比Fas、Fasl和Caspase3的蛋白水平顯著降低,TUNEL染色陽性細(xì)胞數(shù)減少,而中性粒細(xì)胞數(shù)目是增多的。滅活的煙曲霉菌刺激中性粒細(xì)胞4小時(shí)、8小時(shí)、12小時(shí)、16小時(shí)后Mincle的m RNA和蛋白比正常對照組中的表達(dá)高,在8小時(shí)Mincle的m RNA表達(dá)水平最高。TDB處理組和DMSO處理組相比,TDB組Mincle的m RNA表達(dá)增多。TDB預(yù)處理組煙曲霉菌刺激小鼠中性粒細(xì)胞8小時(shí)后,Fas、Fasl和Caspase3的m RNA和蛋白表達(dá)水平與單純的煙曲霉菌刺激相比顯著降低,中性粒細(xì)胞凋亡率下降;Ig G預(yù)處理組煙曲霉菌刺激8小時(shí)后Fas、Fasl和Caspase3的m RNA和蛋白表達(dá)水平與Mincle中和抗體預(yù)處理組相比顯著降低,中性粒細(xì)胞的凋亡率下降。結(jié)論:Mincle參與煙曲霉菌性角膜炎的炎癥反應(yīng)過程,真菌感染后Mincle的表達(dá)上調(diào)同時(shí)中性粒細(xì)胞的浸潤水平和凋亡數(shù)目也是增加的。Mincle還可以通過抑制FAS和FASL的結(jié)合來抑制Caspase3的表達(dá)進(jìn)而抑制嗜中性粒細(xì)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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R772.21

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