PTX3在角膜上皮抗真菌固有免疫阶段作用的研究

发布时间：2018-07-16 21:52

【摘要】：目的:(1)研究PTX3在角膜上皮抗真菌固有免疫中的表达及规律。(2)研究Dectin-1/Syk和MAPKs信号通路是否参与了角膜上皮中PTX3的表达调控。(3)探讨内源性PTX3在角膜上皮抗真菌固有免疫中的作用及其可能的机制。方法:本研究分三部分进行研究。一、分别以真菌性角膜炎患者、真菌性角膜炎大鼠模型和体外培养的人角膜上皮细胞为研究对象,探讨PTX3在真菌感染后的表达规律。采用荧光定量PCR检测PTX3 mRNA的表达、免疫组织化学法或Western Blot检测PTX3蛋白的表达。(1)选择2013年5月到2015年10月在青岛大学附属医院眼科确诊为烟曲霉菌感染并行治疗的角膜炎病人和正常供体的角膜标本。收集病灶角膜的周边角膜上皮组织作为感染组,健康角膜制作角膜移植片后剩余的周边上皮组织作为正常对照。分别检测在角膜烟曲霉菌感染前后PTX3 mRNA和蛋白的表达变化,利用免疫组织化学染色方法对PTX3蛋白在人角膜组织中的表达进行定位。同时,根据角膜溃疡炎症反应程度对样本进行分组(轻度、中度、重度),分析比较PTX3与真菌感染后不同程度炎症反应之间的关系。(2)将50只健康Wistar大鼠随机分为3组:空白对照组、损伤对照组和真菌感染组,刮取角膜上皮检测各组间PTX3在mRNA和蛋白水平的表达差异。(3)在体外培养的永生化人角膜上皮细胞(HCECs)中,用灭活的烟曲霉菌孢子抗原刺激体外培养的HCECs,建立烟曲霉菌感染的体外模型,检测烟曲霉菌刺激不同时间后PTX3在基因和蛋白水平的表达规律。二、选取烟曲霉菌刺激下PTX3基因和蛋白水平表达最显著的时间点(4h和8h),分别用Laminarin(Dectin-1阻断剂)、Piceatannol(Syk抑制剂)、SB203580(p38MAPK抑制剂)、U0126-Et OH(MEK抑制剂)、SP600125(JNK抑制剂)预处理HCECs后,加入灭活烟曲霉菌孢子抗原混悬液,探究Dectin-1/Syk和MAPK信号通路是否参与了PTX3的表达调控。三、研究PTX3在角膜上皮抗烟曲霉菌固有免疫过程中被诱导产生后对炎症的影响。在体外培养的人角膜上皮细胞中,利用PTX3 siRNA预处理HCECs阻断内源性PTX3的产生,再与烟曲霉菌孢子共同培养,Western Blot检测炎症信号通路分子MAPKs蛋白总量和磷酸化水平的变化,荧光定量PCR和酶联免疫吸附试验(ELISA)分别检测终末炎症因子IL-1β在mRNA和蛋白水平的表达情况,了解内源性PTX3影响角膜上皮抗真菌固有免疫的机制。结果:(1)正常人、大鼠角膜上皮和体外培养的角膜上皮细胞可以表达PTX3。轻、中、重组真菌性角膜炎患者角膜上皮中PTX3 mRNA的表达较正常组均显著升高;角膜炎症程度越重,PTX3的表达量越高(p0.001)。烟曲霉菌感染大鼠模型中,角膜上皮PTX3 mRNA和蛋白的表达均在16h达高峰。PTX3在真菌感染组的表达明显高于正常对照组和损伤对照组(p0.05,p0.001)。在体外培养的角膜上皮细胞中,烟曲霉菌孢子刺激可以上调PTX3的表达,其mRNA和蛋白分别在4h和8h达高峰(p0.001)。(2)利用piceatannol、U0126-EtOH、SP600125分别抑制Syk、MEK、JNK可以下调烟曲霉菌诱导的PTX3的产生(p0.001),而laminarin、SB203580分别抑制Dectin-1、p38 MAPK后对烟曲霉菌诱导的PTX3产生无明显影响(p0.05)。(3)siRNA阻断内源性PTX3可以下调角膜上皮细胞在烟曲霉菌刺激时IL-1β的表达(p0.001)、降低MAPK通路中ERK1/2和JNK蛋白的磷酸化水平(p0.001),但对p38 MAPK蛋白的磷酸化水平和MAPKs总蛋白的表达水平无明显影响(p0.05)。结论:PTX3是角膜上皮抗烟曲霉菌免疫的一个重要组成部分,可以反映炎症的程度。Syk、MEK1/2和JNK是调控烟曲霉菌刺激时角膜上皮表达PTX3的靶点。PTX3可以通过促进角膜上皮的炎症反应发挥抗烟曲霉菌的作用。
[Abstract]:Objective: (1) to study the expression and regularity of PTX3 in the antifungal inherent immunity of corneal epithelium. (2) whether the Dectin-1/Syk and MAPKs signaling pathways are involved in the regulation of the expression of PTX3 in the corneal epithelium. (3) to explore the role of endogenous PTX3 in the anti fungal inherent immunity of corneal epithelium and its possible mechanism. Methods: This study was divided into three parts. We studied the expression of PTX3 in the fungal keratitis model and human corneal epithelial cells cultured in vitro respectively. The expression of PTX3 after fungal infection was investigated. The expression of PTX3 mRNA was detected by fluorescence quantitative PCR, and the expression of PTX3 protein was detected by immunohistochemistry or Western Blot. (1) May 2013 The corneal epithelial tissue surrounding the cornea of the focus was collected as an infection group, and the remaining peripheral skin tissue of the cornea was made as normal control. The angle of the cornea was detected in the cornea of the eye department of the Affiliated Hospital of Qiingdao University in October 2015. The expression of PTX3 mRNA and protein in the infection of Aspergillus fumigatus before and after infection of Aspergillus fumigatus, the expression of PTX3 protein in human corneal tissue was located by immunohistochemical staining. Meanwhile, the samples were grouped according to the degree of inflammatory reaction of corneal ulcer (mild, moderate, severe), and the inflammatory response of different degrees after PTX3 and fungal infection was analyzed and compared. (2) 50 healthy Wistar rats were randomly divided into 3 groups: the blank control group, the damage control group and the fungal infection group, the corneal epithelium was scraped to detect the difference in the expression of PTX3 at the level of mRNA and protein. (3) the inactivated Aspergillus fumigatus spores antigen was used to stimulate in vitro culture in vitro. Cultured HCECs, establish an in vitro model of Aspergillus fumigatus infection, detect the expression of PTX3 at gene and protein levels after different times of stimulation of Aspergillus fumigatus. Two, select the most significant time points of PTX3 gene and protein expression (4h and 8h) under the stimulation of Aspergillus fumigatus (4h and 8h), Laminarin (Dectin-1 blocker), Piceatannol (Syk inhibitor), SB20, respectively. 3580 (p38MAPK inhibitor), U0126-Et OH (MEK inhibitor) and SP600125 (JNK inhibitor) pretreated HCECs, adding inactivated Aspergillus fumigatus spores antigen suspension to explore whether Dectin-1/Syk and MAPK signaling pathways were involved in the expression and regulation of PTX3. Three, PTX3 was induced in the inherent immune process of Aspergillus fumigatus in the upper cornea. In human corneal epithelial cells cultured in vitro, PTX3 siRNA pretreated HCECs to block endogenous PTX3 production, and then co culture with Aspergillus fumigatus spores. Western Blot was used to detect the changes in the total amount of MAPKs protein and phosphorylation level of the inflammatory signaling pathway molecules, and the fluorescence determination PCR and enzyme linked immunosorbent assay (ELISA) were detected respectively. The expression of terminal inflammatory factor IL-1 beta at the level of mRNA and protein to understand the mechanism of endogenous PTX3 affecting the antifungal inherent immunity of corneal epithelium. Results: (1) normal people, corneal epithelium and cultured corneal epithelial cells in vitro can express PTX3. light, and the expression of PTX3 mRNA in the corneal epithelium of the patients with recombinant fungal keratitis is more positive. The higher the degree of keratitis, the higher the degree of keratitis, the higher the expression of PTX3 (p0.001). In the rat model of Aspergillus fumigatus infection, the expression of PTX3 mRNA and protein in the corneal epithelium at the peak of 16h was higher than that of the normal control group and the damage control group (P0.05, p0.001). The corneal epithelial cells cultured in vitro were in vitro. The spore stimulation of Aspergillus fumigatus can increase the expression of PTX3, and its mRNA and protein at 4H and 8h peak (p0.001). (2) using piceatannol, U0126-EtOH, SP600125 to inhibit Syk, MEK, JNK can reduce the production of Aspergillus fumigatus induced by Aspergillus fumigatus. There was no significant effect of PTX3 (P0.05). (3) siRNA blocking endogenous PTX3 can down regulate the expression of IL-1 beta in corneal epithelial cells (p0.001) and decrease the phosphorylation level of ERK1/2 and JNK protein in MAPK pathway (p0.001), but there is no significant effect on the phosphorylation level of p38 MAPK protein and the expression level of total protein. PTX3 is an important part of the immunity of corneal epithelia against Aspergillus fumigatus. It can reflect the degree of inflammation of.Syk. MEK1/2 and JNK are the target.PTX3 that regulate the expression of PTX3 in corneal epithelium stimulated by Aspergillus fumigatus. The effect of anti Aspergillus fumigatus can be played by promoting the inflammatory reaction of corneal epithelium.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R772.2

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