阿薩希毛孢子菌體外抗氧化誘導(dǎo)及其生物學(xué)特性研究
發(fā)布時(shí)間：2019-01-04 16:13
【摘要】：研究背景及目的真菌抗氧化應(yīng)激與其感染機(jī)制密切相關(guān),也是近年來的研究熱點(diǎn)。獲得穩(wěn)定的抗氧化菌株是深入研究其抗氧化機(jī)制的前提。阿薩希毛孢子菌Trichosporon asahii(T.asahii)是重要的條件致病菌,其感染機(jī)制的相關(guān)研究匱乏。本實(shí)驗(yàn)研究體外能否用常用氧化劑過氧化氫(hydrogen peroxide,H_2O_2)和亞硫酸氫鈉甲萘醌(menadione sodium bisulfate,MSB)誘導(dǎo)T.asahii產(chǎn)生抗氧化性,以及誘導(dǎo)獲得的抗氧化表型的穩(wěn)定性,并觀察誘導(dǎo)前后菌株的生理學(xué)特性變化。方法1.將T.asahii臨床標(biāo)準(zhǔn)株CBS2479和環(huán)境株CBS8904在H202或MSB濃度逐漸梯級(jí)倍增的YPD液體培養(yǎng)基中傳代,直至無法誘導(dǎo)為止。取誘導(dǎo)終末代菌株在不含氧化劑的YPD液體培養(yǎng)基中連續(xù)傳代10次,進(jìn)行誘導(dǎo)回復(fù)。測(cè)定誘導(dǎo)前、誘導(dǎo)終末代、回復(fù)終末代等不同階段菌株的過氧化氫酶(catalase,CAT)和超氧化物歧化酶(superoxide dismutase,SOD)活性,并應(yīng)用SPSS20.0軟件分析酶學(xué)檢測(cè)數(shù)據(jù)。2.取誘導(dǎo)前、誘導(dǎo)終末代、回復(fù)終末代的菌株行肉眼形態(tài)和光鏡下形態(tài)觀察。3.參照美國臨床和實(shí)驗(yàn)室標(biāo)準(zhǔn)協(xié)會(huì)推薦的M27-A3標(biāo)準(zhǔn)測(cè)定兩種氧化劑誘導(dǎo)前、誘導(dǎo)中期、誘導(dǎo)末期、回復(fù)中期、回復(fù)末期的菌株對(duì)抗真菌藥氟康唑的MIC值和對(duì)氧化劑H_2O_2和MSB的MIC值。結(jié)果1.抗氧化酶活性變化測(cè)定經(jīng)H_2O_2誘導(dǎo)后,CBS2479菌株誘導(dǎo)末期的CAT值、SOD值分別比誘導(dǎo)前升高了約2.15、2.97倍;CBS8904菌株誘導(dǎo)末期的CAT值、SOD值分別比誘導(dǎo)前升高了約1.79、1.79倍。經(jīng)MSB誘導(dǎo)后,CBS2479菌株誘導(dǎo)末期的CAT值、SOD值各比誘導(dǎo)前上升了約3.36、2.68倍;CBS8904菌株誘導(dǎo)末期的CAT值、SOD值分別比誘導(dǎo)前升高了約2.27、2.57倍。經(jīng)H202和MSB誘導(dǎo)后,CBS2479菌株的CAT和SOD活性均顯著升高(P0.05)�；貜�(fù)后的 CAT、SOD 均比誘導(dǎo)前高(P0.05)。CBS2479-H_2O_2、CBS2479-MSB菌株回復(fù)后的CAT比誘導(dǎo)末期下降(P0.05),而SOD則無統(tǒng)計(jì)學(xué)差異。經(jīng)H_2O_2和MSB誘導(dǎo)后,CBS8904菌株的CAT和SOD活性均比誘導(dǎo)前升高(P0.05)。CBS8904-H_2O_2菌株回復(fù)后的SOD比誘導(dǎo)前高(P0.05),但回復(fù)后CAT與誘導(dǎo)前比較無統(tǒng)計(jì)學(xué)差異(P0.05)。CBS8904-MSB菌株回復(fù)后的CAT和SOD值均比誘導(dǎo)前升高(P0.05)。CBS8904-H_2O_2、CBS8904-MSB菌株回復(fù)后的CAT、CBS8904-MSB菌株回復(fù)后的SOD均比誘導(dǎo)末期下降(P0.05),而CBS8904-H_2O_2菌株回復(fù)后的SOD與誘導(dǎo)末期相比無統(tǒng)計(jì)學(xué)差異(P0.05)。2.形態(tài)學(xué)變化誘導(dǎo)后菌落肉眼形態(tài)發(fā)生一定改變;光鏡下可見CBS2479、CBS8904分別由誘導(dǎo)前的菌絲狀、孢子狀變?yōu)檎T導(dǎo)后呈假菌絲與孢子混合狀態(tài)。3.藥物敏感性變化測(cè)定T.asahii分別在H202、MSB濃度梯級(jí)倍增的YPD液體培養(yǎng)基中培養(yǎng),經(jīng)過4次傳代后,CBS2479-H_2O_2、CBS8904-H_2O_2、CBS2479-MSB、CBS8904-MSB菌株對(duì)氟康唑、H_2O_2和MSB的MIC值均為誘導(dǎo)前MIC值的2倍。經(jīng)歷回復(fù)性實(shí)驗(yàn)以后,CBS8904-H_2O_2、CBS8904-MSB菌株的MICH_2O_2均恢復(fù)到誘導(dǎo)前水平,但其余均與誘導(dǎo)第4代的MIC值相同。結(jié)論1.利用濃度梯級(jí)倍增的H202、MSB均能成功誘導(dǎo)出具有抗氧化表型的T.asahii菌株,且該表型相對(duì)穩(wěn)定。2.各菌株菌落誘導(dǎo)后肉眼形態(tài)和鏡下形態(tài)發(fā)生一定改變,且誘導(dǎo)后的菌株光鏡下出現(xiàn)假菌絲與孢子混合狀態(tài)。3.誘導(dǎo)后的菌株對(duì)氟康唑的耐受力較前升高,且誘導(dǎo)后的菌株均對(duì)H_2O_2、MSB的耐受力較前提高。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R379
本文編號(hào)：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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R379
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 張楊梅;李海濤;王聰敏;祝賀;敖俊紅;楊蓉婭;;阿薩希毛孢子菌抗氧化酶活性研究[J];實(shí)用皮膚病學(xué)雜志;2016年02期
2 史鋒;黃宇嘯;李永富;;釀酒酵母抗氧化相關(guān)基因突變體對(duì)黃曲霉毒素B_1的清除作用[J];食品與生物技術(shù)學(xué)報(bào);2012年05期
，本文編號(hào)：2400509
本文鏈接：http://sikaile.net/yixuelunwen/jichuyixue/2400509.html
最近更新
教材專著
