豚鼠真菌性皮肤病模型的建立及致病机理初探

发布时间：2018-03-12 18:33

本文选题：犬真菌性皮肤病　切入点：动物模型　出处：《南京农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：犬真菌性皮肤病是由真菌黏附于犬表皮及附属结构引起的,真菌在附着之后对角质蛋白组织寄生或腐生从而迅速繁殖,最终导致局部皮肤出现脱毛、鳞屑、糜烂等病变。浅层真菌是犬真菌皮肤病的主要病原体,基本为皮肤癣菌,可分为毛癣菌属(如须癣毛癣菌)、表皮癣菌属(如絮状表皮癣菌)和小孢子菌属(如犬小孢子菌、石膏样孢子菌)三个属,马拉色菌等少数酵母菌也可以导致发病。该病在各类型犬只和各个地区均常发生,多为条件致病性疾病,种类多样,治疗期较长,治愈率不高。近年来其发病率的增加可能和宠物临床抗生素及免疫抑制剂使用量过大有关。人类也可能被犬传染上该疾病,在抵抗力低下的特殊人群里该病发生率正在上升。对犬真菌性皮肤病的相关研究是动物医学界目前的科研热点,但是相关工作往往直接使用犬作为实验动物,具有价格高、操作复杂、对操作人员造成人身威胁等缺点,本试验尝试建立实验动物模型模拟犬真菌性皮肤病病变以求改善相关缺点。相关研究多集中于该病的诊断和防治,很少涉及致病机理方面的研究,本试验尝试探索在实验动物机体内免疫系统与病原的相互作用,或能对其致病机理有初步的了解。试验一:须毛癣菌皮肤病实验动物模型的建立为了制作安全、方便、可靠的真菌性皮肤病实验动物模型以替代犬投入相关试验,采用在动物背部接种须毛癣菌菌悬液的方法建立了小鼠和豚鼠两种动物的须毛癣菌皮肤病模型,每间隔1d观察局部皮肤病变症状并评分,在接种真菌后第3d、第13 d、第26 d制作局部皮肤石蜡病理切片。为了了解机体免疫状态对真菌致病的影响,在接种前分别使用地塞米松和黄芪多糖作为免疫抑制剂与免疫加强剂注射动物进行对比。结果显示,全部豚鼠及86.7%的地塞米松组小鼠须毛癣菌再培养呈现阳性,建模成功。须毛癣菌对实验动物皮肤的侵害作用在接种真菌后13 d-17 d逐渐达到最高峰,并在之后逐渐减轻症状。豚鼠的病变症状明显而直观,症状较重,而小鼠的病变症状较浅。黄芪多糖组的感染豚鼠对比免疫抑制组豚鼠皮肤病变症状评分减少。该试验初步证明须毛癣菌实验动物模型是可行的,动物机体的免疫状态对须毛癣菌感染造成影响,感染后半个月左右为病变最严重时期。试验二:体外药敏试验和疗效试验为了确定须毛癣菌皮肤病豚鼠模型应用于抗真菌药物疗效学试验的可行性,先以棋盘微量稀释方法进行了须毛癣菌对两性霉素B、特比萘芬、伊曲康唑以及后两种药物联合用药的体外药敏试验,以肉眼进行评判,再在豚鼠模型上用药进行药效学评估,结果显示联合用药的MIC值最低,须毛癣菌消除率最高,症状总积分减少最多,初步判定药物疗效在动物试验和体外药敏试验中基本一致,该模型可用于相关研究。试验三:相对荧光定量PCR法检测细胞因子为了了解动物机体免疫系统在真菌感染后的变化情况,探究真菌皮肤病致病机理,采取须毛癣菌皮肤病模型豚鼠第3d、第13d、第26d的血液,用相对荧光定量PCR方法检测血液总IL-10、IL-1、TNF-α、IFN-γ四种细胞因子的含量,发现Th2细胞因子IL-10在感染须毛癣菌后第3d升高,提示动物机体在刚受到感染时处于免疫抑制状态,而Th1细胞因子IL-1、TNF-α、IFN-γ分别在第3d、第13 d和第26d达到数值高峰,提示细胞免疫系统迅速作出积极应对抵抗真菌入侵,令病情趋于好转。
[Abstract]:Fungal skin diseases caused by fungal adhesion to canine epidermis and accessory structure, when attached to fungal keratin tissue saprophytic to multiply rapidly, resulting in local skin hair removal, scaling, erosion and other diseases. The shallow fungi were the main pathogens of canine fungal skin diseases, as the basic dermatophyte. Can be divided into the genus Trichophyton (such as Trichophyton mentagrophytes and Epidermophyton) (such as Epidermophyton floccosum) and microsporon (such as Microsporum gypseum, spores) three genus Malassezia yeasts and a few can also cause disease. The disease often occurs in all various types of dogs and various regions, for the conditions of pathogenic disease, species diversity, longer treatment, the cure rate is not high. In recent years the incidence rate increased and pet clinical antibiotic and immune inhibitor use is too large. Humans may also be infected with the disease in dogs In particular, people in low resistance to the disease incidence rate is rising. The related research on fungal skin disease is the current research hotspot of animal medicine, but the work is often used directly in dogs as experimental animal, has a high price, complex operation, the disadvantages of operator personal threats, the this test attempts to establish an experimental animal model of simulated fungal skin disease in order to improve the diagnosis and treatment of defects. The researchers concentrate on the disease, rarely involves the study of pathogenic mechanism of this experiment to explore the interaction of the immune system and pathogens in animal body, or to have a preliminary understanding of the disease the mechanism. Experiment 1: Trichophyton skin disease animal model is built in order to make safe, convenient, fungal skin disease reliable experimental animal model to replace the dogs into the relevant test, recovery The Trichophyton skin disease model mice and guinea pigs of two kinds of animal is established by using the method in the animal back inoculation of Trichophyton mentagrophytes bacterial suspension, each interval of 1D observation of local skin lesions and symptoms score at 3D after inoculation of fungi, thirteenth D, twenty-sixth d to make local skin pathological to affect the immune status. Understanding of the pathogenic fungi, respectively using dexamethasone and astragalus polysaccharide as immunosuppressive agents and immune enhancing agent injection were compared in the animal before inoculation. The results showed that dexamethasone group were Trichophyton mentagrophytes all 86.7% guinea pigs and then cultured positive, successful modeling. Against the effect of Trichophyton mentagrophytes in experimental animal skin after inoculation of fungi 13 D-17 D has reached a peak, and then gradually reduce the symptoms. Disease symptoms in guinea pigs significantly and directly, severe symptoms, and disease symptoms in mice is shallow. The APS group Comparison of immune inhibition groups of guinea pigs infected guinea pig skin lesion symptom score decreased. The results showed that t.mentagrophytes experimental animal model is feasible, the immune status of the animal body's impact on Trichophyton mentagrophytes infection, infection of about half a month after the most serious lesion period. Experiment two: the test of drug sensitivity test in vitro in order to determine the feasibility and efficacy application of t.mentagrophytes skin disease in guinea pig model of antifungal efficacy test, first by the checkerboard microdilution method of terbinafine t.mentagrophytes to amphotericin B, itraconazole, drug sensitivity test in vitro and after two drug combination therapy, evaluation to the naked eye, then the medication in the guinea pig model on pharmacodynamics evaluation the results show that a combination of drugs, the lowest MIC value, t.mentagrophytes elimination rate is the highest, the total symptom score decreased most, the preliminary determination of drug efficacy in animal experiments and in vitro Drug sensitivity test is basically the same, the model can be used for related research. Experiment three: relative cytokine detection by fluorescence quantitative PCR in order to understand the animal immune system changes in fungal infection, pathogenic fungi on skin disease mechanism, take Trichophyton skin disease model of guinea pig 3D, 13D, 26D blood, with detection of IL-10, blood relative fluorescence quantitative PCR method IL-1, TNF- alpha, gamma IFN- content of four kinds of cytokines, Th2 cell factor IL-10 in 3D infection increased the Trichophyton mentagrophytes, suggesting that the animal body on immunosuppression in just the infection, while Th1 cytokines IL-1, TNF- alpha IFN-, gamma after 3D, thirteenth D and 26D to the numerical peak, the cellular immune system quickly and actively respond to fungal invasion, so the condition is getting better.

【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.292

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