本文選題：煙曲霉 + 凋亡��； 參考：《青島大學》2017年碩士論文

【摘要】：目的:煙曲霉是一種條件致病菌,人體依靠肺巨噬細胞清除吸入的煙曲霉孢子。煙曲霉是最普遍的可引起侵襲性曲霉病的病原體,由于許多抗真菌藥物毒性大、效率低和不斷增長的抗藥性使可使用的抗真菌療法變得非常有限。激活煙曲霉內(nèi)生的凋亡成為對抗侵襲性曲霉病和其他真菌引發(fā)疾病的新希望。更多了解凋亡相關基因可以為發(fā)展新的抗真菌藥物提供理論依據(jù)。蛋白激酶A對于多種真菌的生長、形態(tài)及毒性等都有著至關重要的調節(jié)作用。人體依靠氧化壓力清除體內(nèi)萌發(fā)的煙曲霉孢子,而蛋白激酶A是多種真核生物的壓力應答調節(jié)器,pkaR為蛋白激酶A的調節(jié)亞基,因此,研究pkaR基因在氧化壓力下對煙曲霉萌發(fā)孢子凋亡的影響及其調節(jié)機制,具有重要的生物醫(yī)學意義。方法:比較野生株和pkaR基因缺失突變株的萌發(fā)孢子在氧化壓力條件下的凋亡參數(shù),來研究pkaR基因是否參與煙曲霉在氧化壓力下凋亡的調控。培養(yǎng)野生株和pkaR基因缺失突變株的萌發(fā)孢子,選擇雙氧水作為氧化壓力與萌發(fā)孢子孵育。萌發(fā)孢子經(jīng)過氧化壓力刺激后,通過亞甲基藍染色和再生實驗檢測經(jīng)氧化壓力刺激后萌發(fā)孢子活性;DCFH-DA染色檢測在相同氧化壓力條件下野生株和突變株萌發(fā)孢子內(nèi)的ROS產(chǎn)生情況;TUNEL實驗檢測萌發(fā)孢子的DNA斷裂情況,作為判斷萌發(fā)孢子凋亡情況的依據(jù);PI染色檢測經(jīng)氧化壓力作用后萌發(fā)孢子細胞膜的完整性。結果:亞甲基藍實驗結果顯示,經(jīng)氧化壓力處理pkaR基因缺失突變株萌發(fā)孢子的存活率明顯高于野生株;胞內(nèi)ROS含量檢測結果顯示,較低強度的氧化壓力條件下WT株萌發(fā)孢子的ROS產(chǎn)生被促進,較高強度的氧化壓力條件下ROS產(chǎn)生被抑制,與野生株相反突變株在較低氧化壓力條件下ROS產(chǎn)量低,較高氧化壓力條件下ROS產(chǎn)量高,在氧化壓力條件下野生株和突變株萌發(fā)孢子的ROS含量有顯著差異;TUNEL實驗表明,隨著氧化壓力的提高,野生株凋亡的萌發(fā)孢子數(shù)量經(jīng)歷升高再降低的過程,而突變株凋亡的萌發(fā)孢子數(shù)量無顯著變化,野生株與突變株相比凋亡的萌發(fā)孢子數(shù)量有顯著差異;PI實驗表明,在氧化壓力條件下WT株的萌發(fā)孢子細胞膜受到的損傷更大,即壞死細胞更多,與突變株相比對于氧化損傷的抵抗能力更弱。結論:亞甲基藍染色作為一種檢測菌體存活率的方法,可以應用于煙曲霉萌發(fā)孢子活性的檢測中;pkaR基因敲除后突變株的萌發(fā)孢子存活率更高,對于氧化壓力的敏感性降低,說明pkaR基因影響煙曲霉萌發(fā)孢子對于氧化損傷的抵抗;經(jīng)氧化壓力刺激后,野生株與突變株的ROS產(chǎn)量有顯著差異,說明pkaR基因參與調節(jié)煙曲霉萌發(fā)孢子ROS產(chǎn)生;在相同氧化壓力條件下,野生株與突變株凋亡的萌發(fā)孢子數(shù)量有顯著差異,說明pkaR基因參與調控煙曲霉萌發(fā)孢子的凋亡。
[Abstract]:Objective: Aspergillus fumigatus is a conditional pathogen. The human body relies on lung macrophages to remove Aspergillus fumigatus spore. Aspergillus fumigatus is the most common cause of invasive aspergillosis. Because many antifungal drugs are toxic, low efficiency and growing resistance make the use of antifungal therapy very limited. The endogenetic apoptosis of moldy has become a new hope to combat invasive aspergillosis and other fungal diseases. More understanding of apoptosis related genes can provide a theoretical basis for the development of new antifungal agents. Protein kinase A plays a crucial role in the growth, morphology and toxicity of various fungi. The human body relies on oxidative stress. In vivo germination of Aspergillus fumigatus spores, protein kinase A is a pressure response regulator of a variety of eukaryotes and pkaR is a regulatory subunit of protein kinase A. Therefore, it is important to study the effect of pkaR gene on the apoptosis of Aspergillus fumigatus and its regulation mechanism under oxidative stress. Methods: compare wild plant and pkaR gene. The apoptosis parameters of the germinating spores of the mutant strain in the oxidative stress condition were studied to investigate whether the pkaR gene was involved in the regulation of apoptosis under oxidative stress. The germination spores of the wild strain and the pkaR gene deletion mutant were incubated with hydrogen peroxide as the oxidation pressure and the germinated spores. The germinated spores were stimulated by oxidative stress, The activity of germinating spores was detected by methylene blue staining and regeneration. The ROS production in the spores of the wild and mutant strains under the same oxidative stress was detected by DCFH-DA staining. The TUNEL test was used to detect the DNA fracture of the germinated spores as the basis for determining the apoptotic conditions of the germinating spores; PI staining was used to detect the spores. The membrane integrity of germinating spores after oxidative stress was measured. Results: the results of methylene blue test showed that the survival rate of the spores of the pkaR gene deletion mutant was significantly higher than that of the wild strain, and the intracellular ROS content detection results showed that the ROS production of the germinated spores of the WT strain was promoted under the low intensity of oxidative stress. The production of ROS was inhibited under the condition of high pressure, and the yield of ROS was low under the lower oxidation pressure, and the yield of ROS was high under the condition of higher oxidative stress. The ROS content of the spores of the wild and mutant strains was significantly different under the condition of oxidative stress. The TUNEL experiment showed that with the increase of oxidation pressure, the TUNEL experiment showed that the oxidative stress increased, The number of germinating spores in the wild plant was increased and then decreased, while the number of germinating spores in the mutant was not significantly changed. The number of germinated spores in the wild plant and the mutant was significantly different. The PI experiment showed that the membrane of the germinated spores of the WT strain was more damaged, that is, necrotic cells under the condition of oxidative stress. More, compared with the mutant strain, the resistance to oxidative damage is weaker. Conclusion: methylene blue staining as a method to detect the survival rate of mycelium can be applied to the detection of the spore activity of Aspergillus fumigatus. The survival rate of the germinating spores of the mutant strain after pkaR knockout is higher, and the sensitivity of the oxidative stress is reduced, indicating the pkaR base. Due to the resistance to oxidative damage in the germination spores of Aspergillus fumigatus, the ROS yield of the wild strain and the mutant strain was significantly different after the oxidative stress, indicating that the pkaR gene was involved in regulating the ROS production of the germinating spores of Aspergillus fumigatus, and the number of the spores of the wild plants and the mutant strains were significantly different under the same oxidation pressure, indicating the pkaR base. It was involved in the regulation of the apoptosis of spore of Aspergillus fumigatus.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R379

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本文編號：1906462