大鼠烟曲霉菌性角膜炎动物模型的建立及其免疫病理的研究

发布时间：2018-04-24 13:11

本文选题：真菌性角膜炎 + 烟曲霉菌　；参考：《同济大学》2006年硕士论文

【摘要】： 目的：建立大鼠烟曲霉菌性角膜炎动物模型。探讨烟曲霉菌感染角膜组织后角膜局部的免疫病理机制。方法：刮除大鼠角膜上皮层，以右眼为实验眼，在角膜面与自制角膜接触镜之间直接涂抹培养的菌落或滴加不同浓度的孢子悬液：以左眼为对照眼，在角膜面与自制角膜接触镜之间滴加无菌生理盐水。观察病程，行角膜病理切片HE和PAS染色，比较得出较好的建模方法，对病程进行合理分期。采用该方法建立实验早期、中期、后期组及对照组。实验组分别于接种后2天(d)、6d、2周(W)时处死取标本，对照组于接种后3d处死取标本。采用免疫组织化学方法观察角膜组织中中性粒细胞、LC、T淋巴细胞(CD3~+T)及其亚群CD4~+T、CD8~+T淋巴细胞、B淋巴细胞的动态变化。采用逆转录聚合酶链式反应(RT-PCR)，检测IL-6、IL-10、细胞间粘附分子-1(ICAM-1)在不同病程角膜组织中的表达。结果：菌落直接涂抹法能稳定且简便的建立大鼠烟曲霉菌性角膜炎动物模型。在大鼠烟曲霉菌性角膜炎早期(接种后1-3d)，，见角膜水肿、角膜白色溃疡浸润灶形成，PAS染色见菌丝平行或斜形生长于角膜基质前1／3层。免疫组化见多量中性粒细胞及少量CD3~+T、CD4~+T、CD8~+T淋巴细胞和LC浸润，未检测到B淋巴细胞。在中期(接种后4-7d)，角膜黄白溃疡、干燥致密。PAS染色见菌丝溃疡灶位于角膜基质前1／3-2／3层，周围被大量炎症细胞包围，中性粒细胞浸润达高峰。免疫组化见T淋巴细胞浸润增加，其中CD4~+T淋巴细胞多于CD8~+T淋巴细胞。LC亦增多，B淋巴细胞开始出现。在晚期(接种后9-14d)，角膜白斑及大量新生血管长入。病理切片见肉芽肿样改变，PAS染色未见菌丝或孢子。免疫组化见中性粒细胞数目有所减少，T淋巴细胞浸润达高峰，CD4~+T淋巴细胞亦多于CD8~+T淋巴细胞，B淋巴细胞少量表达。对照组角膜IL-6、IL-10、ICAM-1几乎不表达或极微量表达。在实验组角膜中，IL-6早期即大量表达，中期炎症反应高峰时亦达高峰，以后逐渐下降；IL-10在早期表达最高以后逐渐下降；ICAM-1早期开始表达中期达高峰以后逐渐下降。IL-6在晚期组与对照组间无差别(p＞0.05)，余早期组、中期组、对照组间均有统计学意义(p＜0.01)。IL-10、ICAM-1在实验对照组与实验组早、中、晚期组四组间均有统计学意义(p＜0.01)。结论：(1)菌落直接涂抹法是建立大鼠烟曲霉菌性角膜炎动物模型较好的方法。(2)大鼠烟曲霉菌性角膜炎病程早中期可能是大量真菌及中性粒细胞导致了角膜损伤，中后期特别是后期则可能是迟发型超敏反应导致的免疫损伤。(3)IL-6在AFK病程中是一个敏感的炎症因子，能反映局部的炎症反应程度。(4)IL-10在AFK病程中呈逐渐下降趋势，早期达高峰，中晚期逐渐下降。(5)ICAM-1在局部白细胞聚集，调节炎症反应、免疫反应强度中发挥重要作用。
[Abstract]:Objective: to establish an animal model of mycelial keratitis in rats. To investigate the immunopathologic mechanism of cornea after infection with aspergillus fumigatus. Methods: the epithelial layer of cornea of rats was scraped, the right eye was used as the experimental eye, the colony was smeared directly between the corneal surface and the self-made contact lens or the spore suspension of different concentration was added: the left eye was used as the control eye. The sterile saline was added between the corneal surface and the corneal contact lens. The course of the disease was observed, the corneal sections were stained with HE and PAS, and a better modeling method was obtained. This method was used to establish the early, middle and late experimental group and control group. The experimental group was killed 2 days after inoculation and the control group was killed 3 days after inoculation. Immunohistochemical method was used to observe the dynamic changes of CD3 ~ T and its subsets in neutrophils of the cornea, CD4 ~ T, CD8 ~ T lymphocytes and B lymphocytes. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of IL-6, IL-10 and ICAM-1 in different corneal tissues. Results: the method of colony direct smear could be used to establish the animal model of aspergillus fumigatus keratitis. In the early stage (1-3 days after inoculation), corneal edema was observed in rats with tobacco-curly fungal keratitis, and pas staining showed that mycelium grew parallel or oblique in the first third layer of corneal stroma. Immunohistochemistry showed the infiltration of many neutrophils, a small amount of CD3 ~ TnT, CD8 ~ T lymphocytes and LC, but no B lymphocytes were detected. In the middle stage (4-7 days after inoculation, yellow and white corneal ulcer, dry dense. Pas staining showed that the mycelium ulcer focus was in the first 1 / 3 / 2 / 3 layer of corneal stroma, surrounded by a large number of inflammatory cells, neutrophil infiltration reached the peak. Immunohistochemical staining showed an increase in T lymphocyte infiltration, in which CD4 ~ T lymphocytes were more than CD8 ~ T lymphocytes. LC was also increased and B lymphocytes began to appear. At the late stage (9-14 d after inoculation), corneal leukoplakia and a large number of neovascularization grew. Pathological sections showed granulomatous changes and pas staining without mycelium or spores. Immunohistochemical staining showed that the number of neutrophils decreased and the infiltration of T lymphocytes reached the peak. The number of CD4- T lymphocytes was also higher than that of CD8- T lymphocytes. In the control group, the expression of IL-6, IL-10 and ICAM-1 was almost no or very small. In the experimental group, IL-6 was highly expressed at the early stage and reached the peak at the middle stage of inflammatory response. The expression of IL-10 decreased gradually after the peak expression of ICAM-1 in the early stage and reached the peak in the middle stage. There was no difference between the late stage group and the control group (p > 0.05), while the remaining early group and the middle stage group, there was no difference between the late group and the control group. There was significant difference between control group and control group (P < 0.01). IL-10 ICAM-1 had statistical significance between experimental group and experimental group (P < 0.01). ConclusionThe colony direct smear method is a better method to establish rat model of tobacco-curving fungal keratitis. (2) in early and middle stage of the disease, a large number of fungi and neutrophils may cause corneal injury in rats. In the middle and late stage, especially in the late stage, it may be the delayed type hypersensitivity. IL-6 is a sensitive inflammatory factor in the course of AFK, and it can reflect the local inflammatory reaction degree. The IL-10 level decreases gradually in the course of AFK, and reaches the peak at the early stage. ICAM-1 decreased gradually in late stage and played an important role in local leukocyte aggregation, regulation of inflammatory response and the intensity of immune response.
【学位授予单位】：同济大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R-332;R772.21

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