趋化因子在真菌性角膜炎中表达的实验研究

发布时间：2018-06-18 01:44

本文选题：真菌性角膜炎 + 趋化因子　；参考：《青岛大学》2011年博士论文

【摘要】：目的构建C57BL/6小鼠真菌性角膜炎模型并进行评价；通过C3基因敲除小鼠探讨补体系统在真菌性角膜炎发病过程中的潜在作用；研究趋化因子在真菌性角膜炎中的表达及潜在作用。方法第一部分：补体系统在角膜真菌感染中的作用选用近交系C57BL/6小鼠作为研究对象。将三种常见致病性真菌(茄病镰刀菌、烟曲霉菌和白色念珠菌)配制成浓度为108CFU/ml菌液。采用表层镜法构建真菌性角膜炎模型,即在刮除角膜上皮的小鼠角膜表面覆盖由大鼠角膜制成的“表层镜”,在其间注入菌液,睑裂缝合24h后拆除缝线。在角膜接种菌液1d、3d、5d和7d时,观察角膜溃疡形态并进行临床评分。将实验动物分为正常对照组和C3基因敲除C57BL/6小鼠感染组(简称C3-/-组),采用表层镜法构建茄病镰刀菌性角膜炎模型。在角膜接种菌液1d、3d、5d和7d时,裂隙灯显微镜观察角膜大体病理改变特点,并根据角膜混浊的面积、密度和表面的规则性对真菌性角膜炎的严重性进行临床评分和组织病理学检测。第二部分：趋化因子在小鼠角膜真菌感染过程中的表达采用基质注射法法分别构建茄病镰刀菌和烟曲霉菌和白色念珠菌角膜炎模型。基质注射生理盐水作为对照组。在角膜接种菌液6h、12h、24h和48h时,对模型临床评分、真菌负荷、组织病理学进行研究,并通过RT-Q-PCR方法检测CCL1、CCL2、CCL5、CCL11、CCL27、CXCL10、CXCL12和CCR7的mRNA表达水平。结果第一部分裂隙灯显微镜检查显示,茄病镰刀菌表面有有大量的苔被附着,烟曲霉菌溃疡大多呈龛状,白色念珠菌溃疡形态不一。临床评分显示,菌液接种后病情呈加重趋势,第3天时最重,之后病情开始减轻。不同真菌所致角膜大体病理改变的变化趋势基本一致(F=3.065,P=0.103)。大体观察和临床评分显示,C3-/-组茄病镰刀菌感染后1d以内为轻度,第3d发展为中度,第5、7d恶化为重度。真菌培养菌落计数结果显示,C3-/-组茄病镰刀菌感染后3d内可见较多菌落生长,第5、7天菌落减少。正常对照组真菌负荷明显小于C3-/-组(P0.05)组织病理学检查显示,正常对照组角膜溃疡在第3天时最重,基质内可见大量炎性细胞浸润,以多形核粒细胞为主；C3-/-组多形核粒细胞进行性增多。第二部分角膜接种两种菌液后临床评分进行升高。白色念珠菌接种后头12h载菌量进行性升高,之后下降,然后再次升高；烟曲霉菌载菌量进行性下降。CCL2、CCL11、CCL27、CXCL10和CXCL12在接种后有升高,其中以CCL2升高最为明显,CXCL10次之。在白色念珠菌性角膜炎中,CCL2在接种12h达到峰值,而CXCL1O在接种6h即达到峰值。在烟曲霉菌性角膜炎中,CCL2在接种24h达到峰值,而CXCL10是在接种12h达到峰值。CXCL12浓度虽有升高但远低于CXCL10。结论通过表层镜法可以成功建立C57BL/6小鼠常见致病真菌性角膜炎模型。当补体系统功能缺陷时,角膜真菌感染早期天然免疫防御功能明显减弱,角膜容易发生坏死和穿孔,但感染早期角膜炎症反应却较补体功能正常组轻。趋化因子CCL2和CXCL10可能在真菌性角膜炎发病早期发挥重要作用。趋化因子CXCL10和CXCL12可能影响角膜新生血管的发生和发展。
[Abstract]:objective
The C57BL/6 mouse fungal keratitis model was constructed and evaluated. The potential role of the complement system in the pathogenesis of fungal keratitis was explored through C3 knockout mice, and the expression and potential effect of chemokines in fungal keratitis was studied.
Method
Part I: the role of complement system in corneal fungal infection
The C57BL/6 mice were used as the research object. Three common pathogenic fungi (Fusarium Solanum, Aspergillus fumigatus and Candida albicans) were prepared into the concentration of 108CFU/ml bacteria. The epiphytic keratitis model was constructed by the surface mirror method, which covered the "surface mirror" made from the cornea of the rat cornea on the cornea surface of the cornea that scraped the corneal epithelium. In the meantime, the bacterial fluid was injected and the eyelid cleft was sutured after 24h to dismantle the suture. When the cornea was inoculated with 1D, 3D, 5D and 7d, the corneal ulcer morphology was observed and the clinical score was observed. The experimental animals were divided into normal control group and C3 gene knockout C57BL/6 mice infection group (C3-/- group), and the Fusarium keratitis model of Solanum Solanum was constructed by the surface mirror method. At the time of 1D, 3D, 5D and 7d, the characteristics of corneal pathological changes were observed in the slit lamp microscope, and the severity of fungal keratitis was evaluated by clinical score and histopathology according to the area, density and surface regularity of corneal opacities.
The second part: expression of chemokine in mouse corneal fungal infection.
The matrix injection method was used to construct Fusarium Solanum, Aspergillus fumigatus and Candida albicans respectively. Matrix injection of saline was used as the control group. When the cornea was inoculated with 6h, 12h, 24h and 48h, the model clinical score, fungus load, histopathology were studied, and CCL1, CCL2, CCL5, CCL11, CCL27 were detected by RT-Q-PCR method. MRNA expression levels of CXCL10, CXCL12 and CCR7.
Result
Part one
The slit lamp microscopy showed that a large number of moss were attached to the Fusarium Solanum on the surface of Solanum, most of Aspergillus fumigatus ulcers were in niches, and the morphology of Candida albicans was different. The clinical score showed that the disease was aggravated after inoculation, and the condition was the heaviest at third days. The changes of the pathological changes of cornea caused by different fungi were changed. The trend was basically the same (F=3.065, P=0.103). The gross observation and clinical score showed that the infection of Fusarium Solanum in C3-/- group was mild in 1D, the development of 3D was moderate, and the 5,7d deteriorated to severe. The colony count results of fungus culture showed that more colonies could be seen in 3D, and the colony decreased at 5,7 after the infection of Fusarium Solanum in the C3-/- group, and the normal control was the normal control. The group fungal load was less than C3-/- group (P0.05) histopathological examination showed that the corneal ulcer was the heaviest in the normal control group at third days, a large number of inflammatory cells infiltrated in the matrix, and polymorphonuclear granulocyte was mainly in the C3-/- group, and the polymorphonuclear granulocyte in the group of C3-/- was increased.
The second part
The clinical score of the cornea was increased after inoculation of two kinds of bacteria. After inoculation of Candida albicans, the amount of 12h carrying bacteria increased, then decreased, and then increased again; the bacteria carrying amount of Aspergillus fumigatus decreased.CCL2, CCL11, CCL27, CXCL10 and CXCL12 increased after inoculation, among which CCL2 was the most obvious, CXCL10 time. In keratitis, CCL2 reached its peak at 12h inoculation, while CXCL1O reached its peak at 6h. In Aspergillus fumigatus keratitis, CCL2 reached its peak at 24h, while CXCL10 was higher but far lower than CXCL10. in the peak.CXCL12 concentration of 12h.
conclusion
The common pathogenic fungal keratitis model of C57BL/6 mice can be successfully established by the surface mirror method. When the function defect of the complement system, the early natural immune defense function of the corneal fungal infection is obviously weakened, the cornea is prone to necrosis and perforation, but the early keratitis response to the infection is lighter than the complement function normal group. Chemokine CCL2 and CXCL 10 may play an important role in the early stage of fungal keratitis. Chemokines CXCL10 and CXCL12 may affect the occurrence and development of corneal neovascularization.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R772.21

【参考文献】