發(fā)布時(shí)間：2018-07-05 06:19

  本文選題：阿薩希毛孢子菌 + 氧化應(yīng)激�。� 參考：《山西醫(yī)科大學(xué)》2012年碩士論文

【摘要】：研究背景 毛孢子菌屬于半知菌、芽生菌綱、隱球酵母目、隱球酵母科，在自然界中廣泛存在，是皮膚、口腔、指甲的正常寄生菌。它可引起淺部感染，如毛發(fā)、皮膚、指甲的白毛結(jié)節(jié)病和免疫缺陷患者的系統(tǒng)性或播散性感染，主要見于惡性腫瘤、呼吸功能不全、慢性腎功能不全、血液病、糖尿病、肝硬化或艾滋病患者。其主要風(fēng)險(xiǎn)因素有：抗生素、免疫抑制劑、腎上腺皮質(zhì)激素、細(xì)胞毒性藥物的使用，以及各種醫(yī)療操作如機(jī)械通氣、靜脈插管、導(dǎo)尿術(shù)、器官移植、手術(shù)操作或持續(xù)性腹膜透析等[1-4]。目前尚有關(guān)于健康者感染此病原菌導(dǎo)致系統(tǒng)性感染的報(bào)告[5-8]，阿薩希毛孢子菌是其重要致病菌。 阿薩希毛孢子菌（Trichosporon asahii，T.asahii）所致播散性毛孢子菌病的臨床研究中發(fā)現(xiàn)，盡管使用抗真菌藥物，但感染仍持續(xù)存在。由于該菌的致病機(jī)制、毒力基因與耐藥機(jī)制等方面的研究目前尚不清楚，且該菌對(duì)絕大多數(shù)的抗真菌藥均耐藥，一旦該菌在機(jī)體造成了播散性、系統(tǒng)性感染，死亡率很高[9]。因此，探索該菌侵襲及感染機(jī)制，顯得尤為重要。目前，已經(jīng)用于臨床或正在研究的抗真菌藥物主要作用于真菌細(xì)胞膜、細(xì)胞壁、線粒體、影響核酸和蛋白質(zhì)合成及功能[10、11]。在上述藥物中，有的藥物因耐藥或毒副作用在臨床應(yīng)用受到很大限制，個(gè)別新近上市的藥物價(jià)格太高病人難以接受。近年來(lái)，氧化應(yīng)激機(jī)制在抗真菌藥的研究方面受到關(guān)注。比如：白念珠菌的抗菌機(jī)制研究，通過(guò)誘導(dǎo)氧化應(yīng)激反應(yīng)殺傷真菌是目前某些抗真菌藥物的作用機(jī)制之一[12-14]。真菌病原體侵入機(jī)體后，首先面臨的是機(jī)體固有免疫系統(tǒng)的防御，誘導(dǎo)機(jī)體進(jìn)入氧化應(yīng)激狀態(tài)。吞噬細(xì)胞釋放大量活性氧介質(zhì)（ROS），包括過(guò)氧化氫、超氧陰離子、羥自由基、過(guò)氧化物等；ROS和其他細(xì)胞因子共同作用使被吞噬的病原真菌經(jīng)受致死性打擊，病原菌被清除[15]。ROS的損傷作用對(duì)清除病原菌極為重要，而病原菌通過(guò)形態(tài)改變耐受氧化損傷的能力增強(qiáng)可能導(dǎo)致其免疫逃逸。由此看來(lái)，利用ROS和氧化應(yīng)激反應(yīng)可能成為抗真菌的有效途徑之一。 研究目的 本課題應(yīng)用三種氧化劑模擬體內(nèi)氧化應(yīng)激環(huán)境，分別在肉眼、光鏡、掃描電鏡下觀察H2O2、二酰胺和甲萘醌三種氧化劑對(duì)阿薩希毛孢子菌臨床分離株與環(huán)境分離株生長(zhǎng)發(fā)育的影響。 研究方法 1．以臨床分離株（菌株號(hào)：CBS2479）和環(huán)境分離株（菌株號(hào)：CBS8904）T.asahii（均購(gòu)自荷蘭CBS公司）為研究對(duì)象，配制菌懸液，分別接種在加入不同濃度H2O2、二酰胺、甲萘醌的PDA混合培養(yǎng)基上培養(yǎng)5天，肉眼觀察三種氧化劑對(duì)兩株菌菌落生長(zhǎng)情況的影響，并與空白對(duì)照相比較。 2．臨床分離株和環(huán)境分離株T.asahii分別接種在加入三種氧化劑與液體YPD混合培養(yǎng)基中，振蕩培養(yǎng)3天，乳酸酚棉藍(lán)染色觀察，光鏡下觀察形態(tài)變化，生長(zhǎng)發(fā)育情況，并與空白對(duì)照相比較。 3．把臨床分離株和環(huán)境分離株T.asahii分別接種在加入不同濃度三種氧化劑的PDA混合培養(yǎng)基上培養(yǎng)48h，挑取一團(tuán)菌落，制成掃描電鏡標(biāo)本，在掃描電鏡下觀察形態(tài)變化，并與空白對(duì)照相比較。 結(jié)果 1.肉眼觀察，臨床分離株和環(huán)境分離株T.asahii在加入不同濃度梯度三種氧化劑的PDA混合培養(yǎng)基上生長(zhǎng)情況不同，隨氧化劑濃度的增加，菌落直徑減小，當(dāng)氧化劑濃度增大到一定數(shù)值時(shí)，菌株不再生長(zhǎng)，三種氧化劑對(duì)環(huán)境分離株的影響較大，，同一氧化劑相同濃度條件下菌落直徑較臨床分離株小。 2.光鏡觀察，不同濃度的三種氧化劑對(duì)臨床分離株和環(huán)境分離株T.asahii有明顯的影響，與空白對(duì)照相比較，可見大量發(fā)育不全未著色發(fā)亮的小孢子以及細(xì)胞碎片，偶可見胞壁皺縮的較大孢子，幾乎未見菌絲，而空白菌株見許多菌絲；隨培養(yǎng)時(shí)間的延長(zhǎng)，發(fā)育不全的孢子未見明顯變化。 3.掃描電鏡觀察，臨床分離株空白對(duì)照可見大量的菌絲，表面不光滑，有皺褶，直徑約2μm；環(huán)境分離株空白對(duì)照見大量鏈狀孢子、關(guān)節(jié)孢子，表面有絨毛，孢子直徑約3μm。三種氧化劑對(duì)兩株菌形態(tài)均有影響，臨床分離株出現(xiàn)部分菌絲胞壁破壞，胞質(zhì)外溢，菌絲表面皺褶消失、光滑，體積縮小，偶可見整個(gè)菌絲完全被破壞。環(huán)境分離株出現(xiàn)胞壁凹陷、萎縮、破壞，胞質(zhì)外溢，表面絨毛消失。甲萘醌對(duì)兩株菌影響最大，二酰胺次之，H2O2最小。 結(jié)論 氧化劑濃度越高，臨床分離株和環(huán)境分離株T.asahii數(shù)量越少，濃度達(dá)到一定數(shù)值后，兩株菌不再生長(zhǎng)。三種氧化劑影響臨床分離株和環(huán)境分離株T.asahii的生長(zhǎng)發(fā)育，有菌絲狀態(tài)轉(zhuǎn)為孢子狀態(tài)傾向，對(duì)環(huán)境分離株影響更大一些；三種氧化劑中，甲萘醌對(duì)兩株菌作用效果最強(qiáng)，二酰胺次之，H2O2最小。推測(cè)T.asahii內(nèi)存在抗氧化機(jī)制，當(dāng)氧化作用超過(guò)抗氧化作用時(shí)，菌株數(shù)量減少，或不再生長(zhǎng)。
[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 .
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R756

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