阿萨希毛孢子菌体外抗氧化诱导及其生物学特性研究
发布时间：2019-01-04 16:13
【摘要】：研究背景及目的真菌抗氧化应激与其感染机制密切相关,也是近年来的研究热点。获得稳定的抗氧化菌株是深入研究其抗氧化机制的前提。阿萨希毛孢子菌Trichosporon asahii(T.asahii)是重要的条件致病菌,其感染机制的相关研究匮乏。本实验研究体外能否用常用氧化剂过氧化氢(hydrogen peroxide,H_2O_2)和亚硫酸氢钠甲萘醌(menadione sodium bisulfate,MSB)诱导T.asahii产生抗氧化性,以及诱导获得的抗氧化表型的稳定性,并观察诱导前后菌株的生理学特性变化。方法1.将T.asahii临床标准株CBS2479和环境株CBS8904在H202或MSB浓度逐渐梯级倍增的YPD液体培养基中传代,直至无法诱导为止。取诱导终末代菌株在不含氧化剂的YPD液体培养基中连续传代10次,进行诱导回复。测定诱导前、诱导终末代、回复终末代等不同阶段菌株的过氧化氢酶(catalase,CAT)和超氧化物歧化酶(superoxide dismutase,SOD)活性,并应用SPSS20.0软件分析酶学检测数据。2.取诱导前、诱导终末代、回复终末代的菌株行肉眼形态和光镜下形态观察。3.参照美国临床和实验室标准协会推荐的M27-A3标准测定两种氧化剂诱导前、诱导中期、诱导末期、回复中期、回复末期的菌株对抗真菌药氟康唑的MIC值和对氧化剂H_2O_2和MSB的MIC值。结果1.抗氧化酶活性变化测定经H_2O_2诱导后,CBS2479菌株诱导末期的CAT值、SOD值分别比诱导前升高了约2.15、2.97倍;CBS8904菌株诱导末期的CAT值、SOD值分别比诱导前升高了约1.79、1.79倍。经MSB诱导后,CBS2479菌株诱导末期的CAT值、SOD值各比诱导前上升了约3.36、2.68倍;CBS8904菌株诱导末期的CAT值、SOD值分别比诱导前升高了约2.27、2.57倍。经H202和MSB诱导后,CBS2479菌株的CAT和SOD活性均显著升高(P0.05)。回复后的 CAT、SOD 均比诱导前高(P0.05)。CBS2479-H_2O_2、CBS2479-MSB菌株回复后的CAT比诱导末期下降(P0.05),而SOD则无统计学差异。经H_2O_2和MSB诱导后,CBS8904菌株的CAT和SOD活性均比诱导前升高(P0.05)。CBS8904-H_2O_2菌株回复后的SOD比诱导前高(P0.05),但回复后CAT与诱导前比较无统计学差异(P0.05)。CBS8904-MSB菌株回复后的CAT和SOD值均比诱导前升高(P0.05)。CBS8904-H_2O_2、CBS8904-MSB菌株回复后的CAT、CBS8904-MSB菌株回复后的SOD均比诱导末期下降(P0.05),而CBS8904-H_2O_2菌株回复后的SOD与诱导末期相比无统计学差异(P0.05)。2.形态学变化诱导后菌落肉眼形态发生一定改变;光镜下可见CBS2479、CBS8904分别由诱导前的菌丝状、孢子状变为诱导后呈假菌丝与孢子混合状态。3.药物敏感性变化测定T.asahii分别在H202、MSB浓度梯级倍增的YPD液体培养基中培养,经过4次传代后,CBS2479-H_2O_2、CBS8904-H_2O_2、CBS2479-MSB、CBS8904-MSB菌株对氟康唑、H_2O_2和MSB的MIC值均为诱导前MIC值的2倍。经历回复性实验以后,CBS8904-H_2O_2、CBS8904-MSB菌株的MICH_2O_2均恢复到诱导前水平,但其余均与诱导第4代的MIC值相同。结论1.利用浓度梯级倍增的H202、MSB均能成功诱导出具有抗氧化表型的T.asahii菌株,且该表型相对稳定。2.各菌株菌落诱导后肉眼形态和镜下形态发生一定改变,且诱导后的菌株光镜下出现假菌丝与孢子混合状态。3.诱导后的菌株对氟康唑的耐受力较前升高,且诱导后的菌株均对H_2O_2、MSB的耐受力较前提高。
[Abstract]:The research background and the target fungus's anti-oxidative stress are closely related to the mechanism of its infection, and it is a hot spot in recent years. It is the premise to study the anti-oxidation mechanism of the stable antioxidant strain. T. asahi (T. ashaii) is an important condition pathogen, and the related research of its infection mechanism is deficient. The oxidation resistance of T. asaii induced by hydrogen peroxide (H _ 2O _ 2) and sodium bisulfite (MSB) in vitro can be induced in vitro, and the physiological characteristics of the strain before and after induction were observed. Method 1. The T. ashaii clinical standard strain CBS2479 and the environmental strain CBS8904 were passaged in a YPD liquid medium with a gradual step-multiplication of H202 or MSB concentration until it was not induced. and inducing the final generation strain to be continuously passaged for 10 times in a YPD liquid culture medium without an oxidizing agent to induce a response. The activity of catalase (CAT) and superoxide dismutase (SOD) and the activity of superoxide dismutase (SOD) were measured before and after induction, and SPSS10.0 software was used to analyze the enzyme detection data. At the end of induction, the final generation of the strain was induced, and the morphology of the last generation of the strain was observed and the morphology was observed under the light microscope. The MIC values of the two oxidizing agents were determined by reference to the M27-A3 standard recommended by the American Society for Clinical and Laboratory Standards, and the MIC values of the strains against the fungal drug and the MIC of the oxidant H _ 2O _ 2 and the MSB were compared with the strains at the end of the induction, at the end of the induction, in the middle of the response, and the end of the response. Results 1. After H _ 2O _ 2 induction, the CAT value and SOD value of CBS2479 were increased by about 2.15, 2.97 times higher than that of induction, respectively. The CAT and SOD values of CBS8904 were increased by about 1.79 and 1.79 times higher than that of induction. After the induction of MSB, the CAT value at the end of induction of CBS2479 increased by about 3.36, 2.68 times higher than that of induction, and the CAT value at the end of induction of CBS8904 was about 2.27, 2.57 times higher than that before induction. After H202 and MSB induction, the activity of CAT and SOD of CBS2479 strain increased significantly (P0.05). The CAT and SOD were higher than that before induction (P0.05). The CAT of CBS2479-H _ 2O _ 2 and CBS2479-MSB was lower than that of the end of induction (P0.05). After the induction of H _ 2O _ 2 and MSB, the activity of CAT and SOD of CBS8904 strain was higher than that before induction (P0.05). The content of CAT and SOD in CBS8904-H _ 2O _ 2 strain was higher than that before induction (P0.05). The CAT and SOD values of CBS8904-MSB strain after the response were higher than that before induction (P0.05). The CAT and SOD values of CBS8904-H _ 2O _ 2 and CBS8904-MSB strain were higher than that before induction (P0.05). The contents of SOD in CBS8904-H _ 2O _ 2 after the recovery of CBS8904-H _ 2O _ 2 were significantly lower than that of the end-of-induction (P0.05). The morphology of the colonies was changed by morphological changes; CBS2479 and CBS8904 were isolated from the hyphae before and after induction, and the spore-like state was the mixed state of the pseudohyphae and the spores. The MIC values of CBS2479-H _ 2O _ 2, CBS8904-H _ 2O _ 2, CBS2479-MSB and CBS8904-MSB were 2 times the MIC value before induction. After the response experiment, the MICH _ 2O _ 2 of CBS8904-H _ 2O _ 2 and CBS8904-MSB strain recovered to the pre-induction level, but the rest was the same as the MIC value for inducing the 4th generation. Conclusion 1. The T. ashaii strain with an anti-oxidation phenotype was successfully induced by the MSB of the H202, which was multiplied by the concentration step, and the phenotype was relatively stable. After the colonies of each strain, the morphology of the naked eye and the morphology of the mirror were changed, and the mixed state of the pseudohyphae and the spores was observed under the light microscope of the strain. The induced strain of the strain was higher than that before the induction, and the induced strain had higher resistance to H _ 2O _ 2 and the MSB of the strain.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R379
本文编号：2400509
[Abstract]:The research background and the target fungus's anti-oxidative stress are closely related to the mechanism of its infection, and it is a hot spot in recent years. It is the premise to study the anti-oxidation mechanism of the stable antioxidant strain. T. asahi (T. ashaii) is an important condition pathogen, and the related research of its infection mechanism is deficient. The oxidation resistance of T. asaii induced by hydrogen peroxide (H _ 2O _ 2) and sodium bisulfite (MSB) in vitro can be induced in vitro, and the physiological characteristics of the strain before and after induction were observed. Method 1. The T. ashaii clinical standard strain CBS2479 and the environmental strain CBS8904 were passaged in a YPD liquid medium with a gradual step-multiplication of H202 or MSB concentration until it was not induced. and inducing the final generation strain to be continuously passaged for 10 times in a YPD liquid culture medium without an oxidizing agent to induce a response. The activity of catalase (CAT) and superoxide dismutase (SOD) and the activity of superoxide dismutase (SOD) were measured before and after induction, and SPSS10.0 software was used to analyze the enzyme detection data. At the end of induction, the final generation of the strain was induced, and the morphology of the last generation of the strain was observed and the morphology was observed under the light microscope. The MIC values of the two oxidizing agents were determined by reference to the M27-A3 standard recommended by the American Society for Clinical and Laboratory Standards, and the MIC values of the strains against the fungal drug and the MIC of the oxidant H _ 2O _ 2 and the MSB were compared with the strains at the end of the induction, at the end of the induction, in the middle of the response, and the end of the response. Results 1. After H _ 2O _ 2 induction, the CAT value and SOD value of CBS2479 were increased by about 2.15, 2.97 times higher than that of induction, respectively. The CAT and SOD values of CBS8904 were increased by about 1.79 and 1.79 times higher than that of induction. After the induction of MSB, the CAT value at the end of induction of CBS2479 increased by about 3.36, 2.68 times higher than that of induction, and the CAT value at the end of induction of CBS8904 was about 2.27, 2.57 times higher than that before induction. After H202 and MSB induction, the activity of CAT and SOD of CBS2479 strain increased significantly (P0.05). The CAT and SOD were higher than that before induction (P0.05). The CAT of CBS2479-H _ 2O _ 2 and CBS2479-MSB was lower than that of the end of induction (P0.05). After the induction of H _ 2O _ 2 and MSB, the activity of CAT and SOD of CBS8904 strain was higher than that before induction (P0.05). The content of CAT and SOD in CBS8904-H _ 2O _ 2 strain was higher than that before induction (P0.05). The CAT and SOD values of CBS8904-MSB strain after the response were higher than that before induction (P0.05). The CAT and SOD values of CBS8904-H _ 2O _ 2 and CBS8904-MSB strain were higher than that before induction (P0.05). The contents of SOD in CBS8904-H _ 2O _ 2 after the recovery of CBS8904-H _ 2O _ 2 were significantly lower than that of the end-of-induction (P0.05). The morphology of the colonies was changed by morphological changes; CBS2479 and CBS8904 were isolated from the hyphae before and after induction, and the spore-like state was the mixed state of the pseudohyphae and the spores. The MIC values of CBS2479-H _ 2O _ 2, CBS8904-H _ 2O _ 2, CBS2479-MSB and CBS8904-MSB were 2 times the MIC value before induction. After the response experiment, the MICH _ 2O _ 2 of CBS8904-H _ 2O _ 2 and CBS8904-MSB strain recovered to the pre-induction level, but the rest was the same as the MIC value for inducing the 4th generation. Conclusion 1. The T. ashaii strain with an anti-oxidation phenotype was successfully induced by the MSB of the H202, which was multiplied by the concentration step, and the phenotype was relatively stable. After the colonies of each strain, the morphology of the naked eye and the morphology of the mirror were changed, and the mixed state of the pseudohyphae and the spores was observed under the light microscope of the strain. The induced strain of the strain was higher than that before the induction, and the induced strain had higher resistance to H _ 2O _ 2 and the MSB of the strain.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R379
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相关期刊论文 前2条
1 张杨梅;李海涛;王聪敏;祝贺;敖俊红;杨蓉娅;;阿萨希毛孢子菌抗氧化酶活性研究[J];实用皮肤病学杂志;2016年02期
2 史锋;黄宇啸;李永富;;酿酒酵母抗氧化相关基因突变体对黄曲霉毒素B_1的清除作用[J];食品与生物技术学报;2012年05期
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