三种氧化剂对阿萨希毛孢子菌生长发育影响的研究

发布时间：2018-07-05 06:19

本文选题：阿萨希毛孢子菌 + 氧化应激　；参考：《山西医科大学》2012年硕士论文

【摘要】：研究背景毛孢子菌属于半知菌、芽生菌纲、隐球酵母目、隐球酵母科，在自然界中广泛存在，是皮肤、口腔、指甲的正常寄生菌。它可引起浅部感染，如毛发、皮肤、指甲的白毛结节病和免疫缺陷患者的系统性或播散性感染，主要见于恶性肿瘤、呼吸功能不全、慢性肾功能不全、血液病、糖尿病、肝硬化或艾滋病患者。其主要风险因素有：抗生素、免疫抑制剂、肾上腺皮质激素、细胞毒性药物的使用，以及各种医疗操作如机械通气、静脉插管、导尿术、器官移植、手术操作或持续性腹膜透析等[1-4]。目前尚有关于健康者感染此病原菌导致系统性感染的报告[5-8]，阿萨希毛孢子菌是其重要致病菌。阿萨希毛孢子菌（Trichosporon asahii，T.asahii）所致播散性毛孢子菌病的临床研究中发现，尽管使用抗真菌药物，但感染仍持续存在。由于该菌的致病机制、毒力基因与耐药机制等方面的研究目前尚不清楚，且该菌对绝大多数的抗真菌药均耐药，一旦该菌在机体造成了播散性、系统性感染，死亡率很高[9]。因此，探索该菌侵袭及感染机制，显得尤为重要。目前，已经用于临床或正在研究的抗真菌药物主要作用于真菌细胞膜、细胞壁、线粒体、影响核酸和蛋白质合成及功能[10、11]。在上述药物中，有的药物因耐药或毒副作用在临床应用受到很大限制，个别新近上市的药物价格太高病人难以接受。近年来，氧化应激机制在抗真菌药的研究方面受到关注。比如：白念珠菌的抗菌机制研究，通过诱导氧化应激反应杀伤真菌是目前某些抗真菌药物的作用机制之一[12-14]。真菌病原体侵入机体后，首先面临的是机体固有免疫系统的防御，诱导机体进入氧化应激状态。吞噬细胞释放大量活性氧介质（ROS），包括过氧化氢、超氧阴离子、羟自由基、过氧化物等；ROS和其他细胞因子共同作用使被吞噬的病原真菌经受致死性打击，病原菌被清除[15]。ROS的损伤作用对清除病原菌极为重要，而病原菌通过形态改变耐受氧化损伤的能力增强可能导致其免疫逃逸。由此看来，利用ROS和氧化应激反应可能成为抗真菌的有效途径之一。研究目的本课题应用三种氧化剂模拟体内氧化应激环境，分别在肉眼、光镜、扫描电镜下观察H2O2、二酰胺和甲萘醌三种氧化剂对阿萨希毛孢子菌临床分离株与环境分离株生长发育的影响。研究方法 1．以临床分离株（菌株号：CBS2479）和环境分离株（菌株号：CBS8904）T.asahii（均购自荷兰CBS公司）为研究对象，配制菌悬液，分别接种在加入不同浓度H2O2、二酰胺、甲萘醌的PDA混合培养基上培养5天，肉眼观察三种氧化剂对两株菌菌落生长情况的影响，并与空白对照相比较。 2．临床分离株和环境分离株T.asahii分别接种在加入三种氧化剂与液体YPD混合培养基中，振荡培养3天，乳酸酚棉蓝染色观察，光镜下观察形态变化，生长发育情况，并与空白对照相比较。 3．把临床分离株和环境分离株T.asahii分别接种在加入不同浓度三种氧化剂的PDA混合培养基上培养48h，挑取一团菌落，制成扫描电镜标本，在扫描电镜下观察形态变化，并与空白对照相比较。结果 1.肉眼观察，临床分离株和环境分离株T.asahii在加入不同浓度梯度三种氧化剂的PDA混合培养基上生长情况不同，随氧化剂浓度的增加，菌落直径减小，当氧化剂浓度增大到一定数值时，菌株不再生长，三种氧化剂对环境分离株的影响较大，，同一氧化剂相同浓度条件下菌落直径较临床分离株小。 2.光镜观察，不同浓度的三种氧化剂对临床分离株和环境分离株T.asahii有明显的影响，与空白对照相比较，可见大量发育不全未着色发亮的小孢子以及细胞碎片，偶可见胞壁皱缩的较大孢子，几乎未见菌丝，而空白菌株见许多菌丝；随培养时间的延长，发育不全的孢子未见明显变化。 3.扫描电镜观察，临床分离株空白对照可见大量的菌丝，表面不光滑，有皱褶，直径约2μm；环境分离株空白对照见大量链状孢子、关节孢子，表面有绒毛，孢子直径约3μm。三种氧化剂对两株菌形态均有影响，临床分离株出现部分菌丝胞壁破坏，胞质外溢，菌丝表面皱褶消失、光滑，体积缩小，偶可见整个菌丝完全被破坏。环境分离株出现胞壁凹陷、萎缩、破坏，胞质外溢，表面绒毛消失。甲萘醌对两株菌影响最大，二酰胺次之，H2O2最小。结论氧化剂浓度越高，临床分离株和环境分离株T.asahii数量越少，浓度达到一定数值后，两株菌不再生长。三种氧化剂影响临床分离株和环境分离株T.asahii的生长发育，有菌丝状态转为孢子状态倾向，对环境分离株影响更大一些；三种氧化剂中，甲萘醌对两株菌作用效果最强，二酰胺次之，H2O2最小。推测T.asahii内存在抗氧化机制，当氧化作用超过抗氧化作用时，菌株数量减少，或不再生长。
[Abstract]:Background of the study
The present invention is one of the main risk factors : antibiotics , immunosuppressive agents , adrenocorticoids , cytotoxic drugs , and various medical operations such as mechanical ventilation , venous catheterization , urinary catheterization , organ transplantation , surgical operation or continuous peritoneal dialysis .
It is found that although antifungal drugs are used , the infection persists despite the use of antifungal agents , which are resistant to most of the antifungal agents . Once the bacteria are disseminated in the organism , systemic infection , mortality is very high . Therefore , it is very important to explore the mechanism of the invasion and infection of the fungus . At present , the antifungal drugs which have been used in clinic or in the study mainly act on the cell membrane , cell wall , mitochondria , affect nucleic acid and protein synthesis and function , and the drug price is too high . In recent years , the mechanism of oxidative stress is very difficult to accept . In recent years , the mechanism of oxidative stress is the defense of the innate immune system and induces the organism to enter oxidative stress state .
The co - action of ROS and other cytokines causes the ingested pathogenic fungi to be subjected to a fatal blow and the pathogenic bacteria are removed . ROS and oxidative stress reaction may become one of the effective ways of antifungal agents .
Purpose of study
In this study , three kinds of oxidant were used to simulate the oxidative stress environment in vivo . The effects of three kinds of oxidant such as H2O2 , diamide and menadione on the growth and development of the isolated plant were observed under the naked eye , light microscope and scanning electron microscope .
Research Methods
1 . A clinical isolate ( strain No . CBS2479 ) and an environmental isolate ( strain No . CBS8904 ) T . asahii ( all from CBS Corporation of the Netherlands ) were used as the research objects , and the suspensions were prepared and inoculated on PDA mixed culture medium with different concentrations of H2O2 , diamide and menadione for 5 days . The effects of three oxidants on the growth of two strains of bacteria were observed by naked eyes and compared with the blank control .
2 . T . asahii , a clinical isolate and an environmental isolate , were inoculated in a mixed culture medium containing three kinds of oxidant and liquid YPD respectively . After shaking culture for 3 days , the blue stain of lactic acid phenol cotton was observed . The morphological changes , growth and development were observed under the light microscope , and compared with the blank control .
3 . T . asahii isolated from clinical isolates and environmental isolates were inoculated on PDA mixed culture medium with different concentrations of three oxidants for 48 hours , then a group of colonies were picked up , and the morphological changes were observed under scanning electron microscope and compared with blank control .
Results
1 . It was observed that T . asahii of clinical isolates and environmental isolates were grown on PDA mixed culture medium with different concentration gradient . As the concentration of oxidant increased , the diameter of colony decreased , and when the concentration of oxidant increased to a certain value , the strain no longer grows , and the three kinds of oxidant have great influence on environment separation plant , and the colony diameter under the same concentration condition of same oxidant is smaller than that of clinical isolates .
2 . Under light microscope , the different concentrations of three kinds of oxidant had obvious effect on T . asahii of clinical isolates and environmental isolates . Compared with blank control , there was a large number of microspores and cell fragments which were not colored and shiny .
With the increase of culture time , no obvious change was found in the development of the spores .
3 . Scanning electron microscope ( SEM ) showed that there were a lot of mycelium in the blank control of clinical isolates , the surface was not smooth , there were wrinkles , and the diameter was about 2 渭m .
There were a large number of chain spores , joint spores , villi on the surface , and about 3 渭m in diameter . Three kinds of oxidizing agents had an influence on the morphology of the two strains . The clinical isolates appeared to be destroyed by the wall of the mycelium , the cytoplasm was reduced , and the whole mycelium was completely destroyed .
Conclusion
The higher the concentration of oxidant , the lower the number of T . asahii in clinical isolates and environmental isolates , and no longer growth of the two strains . The three oxidants affected the growth and development of T . asahii in clinical isolates and environmental isolates .
Among the three kinds of oxidants , menadione has the strongest effect on two strains , the second is the second , H2O2 is the minimum . It is speculated that there is antioxidant mechanism in T . asahii , and the number of strains decreases or no longer grows when the oxidation effect exceeds the antioxidant effect .
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R756

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