利用不同免疫状态小鼠初步探讨真菌性角膜炎的发病机制

发布时间：2018-05-05 20:32

本文选题：真菌性角膜炎 + 发病机制　；参考：《青岛大学》2012年硕士论文

【摘要】：目的本课题组前期利用免疫活性小鼠研究发现特异性免疫在真菌性角膜炎(Fungal Keratitis; FK)发病和转归中发挥重要作用。本研究利用免疫缺陷小鼠进一步探讨免疫反应在真菌性角膜炎发病和转归中的作用。方法选用6-8周龄的正常Balb/c鼠和具有相同遗传背景及相同周龄的裸鼠(T细胞缺陷),SCID鼠(T、B细胞缺陷)作为研究对象,采用基质注射方法分别注射1.0μ1浓度为1×108CFU/ml的白色念珠菌孢子或烟曲霉菌孢子诱导白色念珠菌性角膜炎或烟曲霉菌性角膜炎。在感染后裂隙灯连续观察小鼠角膜临床特征变化,根据角膜病变深度、病灶范围、及表面规则性进行临床评分。在感染后1、3、7和14天取小鼠眼球进行病理学检测,用PAS和HE染色分别观察小鼠角膜组织中真菌菌丝的生长和炎性细胞的浸润；用CFU计数角膜组织中活菌数量的变化；ELISA检测血浆和角膜组织匀浆液中细胞因子IL17、IFNy和IL10的变化；流式细胞分析各组小鼠脾脏中CD19+和IL10+双阳性细胞的动态变化。结果Balb/c鼠和SCID鼠的生理盐水对照组发生极轻度的炎症,其感染组都发生典型的白色念珠菌性角膜炎或烟曲霉菌性角膜炎,两者平均疾病分数都在感染后第5天达到最高随后逐渐降低,并且病理切片染色和CFU检测都显示在感染后1天和3天时其角膜组织中有大量真菌菌丝生长及大量炎性细胞浸润,在感染后14天时其角膜组织已无明显真菌生长,炎症趋向恢复。而相比于Balb/c鼠和SCID鼠发生典型的FK,同样进行真菌孢子注射的裸鼠则不发生白念菌性角膜炎或烟曲霉菌性角膜炎,仅表现为轻度炎症(与正常Balb/c鼠的生理盐水对照组类似),至第7天已恢复正常；并且其角膜组织活菌数在感染后第1天时已明显减少,至第5天已检测不到。Balb/c鼠和SCID鼠生理盐水对照组的皿浆和角膜匀浆液中IL17和IFN7表现为较低水平且随时间无明显变化,但裸鼠感染组中IL17都有早期升高并逐渐回落的趋势,IFNy则都表现为感染中后期升高趋势。而相比于Balb/c鼠和SCID鼠的FK组,裸鼠相应FK组的血浆和角膜匀浆液中IL17和IFN7都表现为较低水平且无明显变化；而IL10则表现为早期较高水平的升高并逐渐回落趋势；脾脏流式检测提示IL10由CD19+细胞分泌。在Balb/c鼠FK组中IL10表现为早期较低水平的升高并逐渐回落趋势,在SCID鼠FK组中检测不到IL10。结论在不同动物诱导FK后对不同细胞因子的动态检测提示：由非特异性免疫细胞产生的IL17在真菌性角膜炎的发病中发挥重要作用;由B细胞分泌的IL10在抑制真菌性角膜炎的发病中发挥关键作用；由非特异性免疫细胞产生的IFNγ在真菌性角膜炎的转归中发挥重要作用。白色念珠菌性角膜炎和烟曲霉菌性角膜炎发病和转归中关键细胞因子的变化趋势大体类似。
[Abstract]:Objective to investigate the role of specific immunity in the pathogenesis and prognosis of fungal keratitis (FK) in mice. The purpose of this study was to investigate the role of immune response in the pathogenesis and outcome of fungal keratitis in immunodeficient mice. Methods normal Balb/c mice aged 6-8 weeks and nude mice with the same genetic background and the same age were selected as the study subjects. Candida albicans or aspergillus fumigatus spores were injected with 1.0 渭 1 concentration of 1 脳 108CFU/ml to induce Candida albicans keratitis or aspergillus fumigatus keratitis respectively. The clinical features of mouse cornea were continuously observed by slit lamp after infection, and the clinical score was evaluated according to the depth of corneal lesion, the extent of lesion and the regularity of surface. The mycelium growth and inflammatory cell infiltration in the cornea of mice were observed by PAS and HE staining on the 7th and 14th day after infection. The changes of the number of living bacteria in corneal tissue were counted by CFU. The changes of cytokines IL17, IFNy and IL10 in plasma and corneal homogenate were detected by Elisa, and the dynamic changes of CD19 and IL10 double positive cells in spleen of each group were analyzed by flow cytometry. Results mild inflammation occurred in the control group of normal saline in Balb/c and SCID mice, and typical candida albicans keratitis or mycosis fumigatus keratitis occurred in the infected group. The mean disease scores of both groups reached the highest level on the 5th day after infection and then decreased gradually. The pathological section staining and CFU detection showed that there were a large number of fungal hyphae growth and inflammatory cell infiltration in corneal tissue on the 1st and 3rd day after infection. At 14 days after infection, there was no obvious fungal growth in corneal tissue and inflammation tended to recover. However, compared with the typical FKs in Balb/c and SCID mice, the same fungal spores injected in nude mice did not develop albicans keratitis or fumigatus keratitis. There was only mild inflammation (similar to the normal saline control group in Balb/c mice, which had returned to normal on the 7th day, and the number of living bacteria in the cornea decreased significantly on the first day after infection. On the 5th day, the IL17 and IFN7 levels in the dish plasma and cornea homogenate of the control group of normal saline in the Balb / c mice and SCID mice were not detected to be lower and had no obvious change with time. However, in nude mice infected group, IL17 increased in early stage and decreased gradually, while IFNy showed an increasing trend in middle and late stage of infection. Compared with the FK group of Balb/c and SCID mice, the IL17 and IFN7 in plasma and cornea homogenate of nude mice showed lower level and no significant change, while IL10 showed a higher level in the early stage and gradually decreased. Splenic flow cytometry showed that IL10 was secreted by CD19 cells. In the FK group of Balb/c rats, the level of IL10 increased at the early stage and decreased gradually, but IL10 was not detected in the FK group of SCID mice. Conclusion the dynamic detection of different cytokines after FK induction in different animals suggests that IL17 produced by non-specific immune cells plays an important role in the pathogenesis of fungal keratitis. IL10 secreted by B cells plays a key role in inhibiting the pathogenesis of fungal keratitis and IFN 纬 produced by non-specific immune cells plays an important role in the outcome of fungal keratitis. The changes of key cytokines in the pathogenesis and outcome of Candida albicans keratitis and mycelium fumigatus keratitis were similar.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R772.21

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