两种地霉对体外健康甲板的侵袭力及胞外酶普的实验性研究

发布时间：2018-06-24 08:13

本文选题：白地霉 + 林生地霉　；参考：《河北医科大学》2012年硕士论文

【摘要】：目的：本实验通过建立真菌透甲模型，探讨林生地霉、白地霉在体外能否侵犯健康甲板，并通过测定透甲时间来比较两种地霉和红色毛癣菌的侵袭作用，同时采用扫描电镜微观观察受侵甲组织的情况。检测两种地霉和红色毛癣菌的细胞外分泌酶活性，比较三者的异同性，结合第一部分结果进行分析，讨论真菌胞外酶与对宿主的侵袭作用的关系。林生地霉（Geotrichum silvicola）为新近发现的一种罕见地霉[1]，我科相继从一例脓癣患儿皮损中和一例中毒性表皮坏死松解症患者的血液中分离得到林生地霉[2,3]，首次证明林生地霉能够侵犯人类。白地霉(Geotrichum candidum)是地霉属的经典代表菌种，为条件性致病菌，以支气管感染最多见。至今未有地霉属真菌侵犯指甲的相关病例报道。红色毛癣菌（Trichophytonrubrum）为亲人性皮肤癣菌，主要侵犯皮肤、指（趾）甲，是我国甲真菌病优势致病菌[4.5]，此试验中红色毛癣菌作为阳性对照菌。方法：林生地霉为河北医科大学第四医院真菌室保存，由中国科学院微生物研究所鉴定。白地霉标准菌株购自中国科学院微生物研究所菌种保藏中心。红色毛癣菌购自北京医科大学附属第一医院真菌实验室。三株真菌均经过复苏培养及形态学和生化鉴定。1.建立真菌透甲模型，分为林生地霉组、白地霉组和红色毛癣菌组，，其中红色毛癣菌组设定为阳性对照组，各组均包括1个阴性对照模型（即不加任何菌种）和1个阳性对照模型（将菌悬液直接加入液体培养基中），观察并记录各组透甲时间，采用统计学方法比较是否存在差异性。2.采用扫描电镜观察受侵甲板的微观变化及菌丝和孢子的形态。3.通过半定量API ZYM细胞外酶活性系统(法国梅里埃生物公司)测定出林生地霉、白地霉及红色毛癣菌的19种胞外酶，包括四种酯酶、五种蛋白酶、二种水解酶和八种糖发酵酶。结果：1.本实验中，三种真菌菌株均能透过甲板模型，其中林生地霉的透甲时间为(15.07±3.28)d,白地霉的透甲时间为(14.50±1.67)d，红色毛癣菌的透甲时间为(6.76±1.72)d。林生地霉和白地霉的透甲时间均明显长于红色毛癣菌（P＜0.05），林生地霉和白地霉的透甲时间无统计学差异（P＞0.05）。2.扫描电镜观察：三株菌株均有菌丝从角化细胞缝隙及细胞中央穿过；3. API-ZYM试剂盒测定结果显示：(1)红色毛癣菌菌株碱性磷酸酶、胰凝乳蛋白酶及多数糖发酵酶活性明显高于林生地霉和白地霉；(2)白地霉和林生地霉的胞外酶谱大致相同，二株菌株具有很高的同源性；(3)三株真菌均含有的胞外酶有：酯酶C4、类脂酯酶C8、白氨酸芳氨酶、缬氨酸芳氨酶、酸性磷酸酶、萘酚-AS-BI-磷酸水解酶。结论：1.林生地霉和白地霉均能穿透甲板，但透甲时间明显长于红色毛癣菌；2.扫描电镜观察：林生地霉、白地霉及红色毛癣菌侵袭的甲板均可观察到有菌丝穿过甲板，菌丝多从角化形成细胞边缘间隙中穿过，部分菌丝直接从角化细胞中央穿出；3.白地霉和林生地霉的胞外酶谱大致相同；4.红色毛癣菌大部分胞外酶活性明显高于林生地霉和白地霉,碱性磷酸酶、胰凝乳蛋白酶及糖发酵酶可能是分解破坏甲板组织的主要作用酶。
[Abstract]:Objective: by establishing a model of fungal penetration in this experiment, we explored the invasion of the health decks in vitro, and by measuring the penetration time to compare the invasion of two kinds of ground molds and Trichophyton rubrum by measuring the time of penetration. At the same time, scanning electron microscopy was used to microscopic observation of the tissues of the invaded armour. The cells of two kinds of ground molds and Trichophyton rubrum were detected. Exocrine activity, comparing the similarities and differences between the three, analyzed the relationship between the fungal extracellular enzyme and the invasion of the host with the results of the first part. Geotrichum silvicola is a newly discovered rare earth mould, [1], and one successively from a case of psoriasis and one case of toxic epidermal necrosis. [2,3] is isolated from the blood of the people, and it is the first time to prove that the Rhizopus of the forest is an invasion of human. Geotrichum candidum is the classic representative of the genus Rhizopus. It is a conditioned pathogen and the most common case is bronchus infection. It is a dermatomycosis of the family members, which mainly invades the skin and refers to the [4.5], the dominant pathogen of onychomycosis in China. In this experiment, Trichophyton rubrum was used as a positive control bacterium.
Methods: Lin Sheng Di mildew was preserved in the fungal room of Fourth Hospital of Hebei Medical University. The standard strain of mildew was bought from the center of bacterial species preservation in the Institute of Microbiology of China Academy of Sciences. Trichophyton red fungus was purchased from the first hospital of Beijing Medical University, the first hospital of the true bacteria laboratory. Three strains of fungi were recovered and cultivated. The morphological and biochemical identification of.1. established a fungal penetration model, which was divided into two groups: forest ground mold, white fungus and Trichophyton rubrum. The Trichophyton red group was set as a positive control group. All the groups included 1 negative control models (that is, without any strains) and 1 positive control models (adding the bacterial suspension directly into the liquid medium). The time of penetration in each group was recorded, and statistical method was used to compare the difference of.2. by scanning electron microscope to observe the microscopic changes of the decks and the morphology of the mycelium and spores of the hyphae and spores.3. by the semi quantitative API ZYM cell outer enzyme activity system (France myrarie biological company) to determine 19 extracellular enzymes of the fungi of the ground molds, mildew and Trichophyton rubrum. It consists of four esterases, five proteases, two hydrolases and eight kinds of sugar fermentation enzymes.
Results: 1. in the 1. experiments, three fungal strains were able to penetrate the deck model, in which the time for the penetration of the fungi was (15.07 + 3.28) d, and the time for the penetration was (14.50 + 1.67) d, and the time for the penetration of Trichophyton rubre was (6.76 + 1.72) d. and the time of penetration was significantly longer than that of Trichophyton rubrum (P < 0.05). There was no statistical difference between the time and the time of penetration (P > 0.05).2. scanning electron microscopy. The three strains all had mycelia from the crevice of keratinized cells and the center of cells. The results of 3. API-ZYM kit showed that (1) the activity of alkaline phosphatase, chymotrypsin and most sugar fermentase activity of Trichophyton rubrum strain was obviously higher than that of forest ground mould And white earth molds; (2) the extracellular enzyme spectrum of Candida alba and phylum Lin was approximately the same, and two strains had high homology; (3) three strains of fungi all contained extracellular enzymes: esterase C4, lipoid esterase C8, alanine aromatase, valine aromatase, acid phosphatase, naphthol -AS-BI- phosphoric acid hydrolase.
Conclusion: 1. the decks can penetrate the deck, but the time of penetration is obviously longer than that of Trichophyton rubrum. 2. scanning electron microscope observation: the decks of mycelia, mycelia and Trichophyton rubrum can be observed through the decks, and the mycelium passes through the diagonalization of the edge of the cell edge, and some mycelium is directly from the keratinocyte The extracellular enzyme spectrum of 3. mildew and ground mildew was approximately the same. 4. most of the extracellular enzyme activity of Trichophyton rubrum was significantly higher than that of fungi and mildew. Alkaline phosphatase, chymotrypsin and sugar fermentase may be the main enzymes that decompose the decks.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R756

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