pkaR基因对烟曲霉萌发孢子凋亡的影响

发布时间：2018-05-18 15:54

本文选题：烟曲霉 + 凋亡　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:烟曲霉是一种条件致病菌,人体依靠肺巨噬细胞清除吸入的烟曲霉孢子。烟曲霉是最普遍的可引起侵袭性曲霉病的病原体,由于许多抗真菌药物毒性大、效率低和不断增长的抗药性使可使用的抗真菌疗法变得非常有限。激活烟曲霉内生的凋亡成为对抗侵袭性曲霉病和其他真菌引发疾病的新希望。更多了解凋亡相关基因可以为发展新的抗真菌药物提供理论依据。蛋白激酶A对于多种真菌的生长、形态及毒性等都有着至关重要的调节作用。人体依靠氧化压力清除体内萌发的烟曲霉孢子,而蛋白激酶A是多种真核生物的压力应答调节器,pkaR为蛋白激酶A的调节亚基,因此,研究pkaR基因在氧化压力下对烟曲霉萌发孢子凋亡的影响及其调节机制,具有重要的生物医学意义。方法:比较野生株和pkaR基因缺失突变株的萌发孢子在氧化压力条件下的凋亡参数,来研究pkaR基因是否参与烟曲霉在氧化压力下凋亡的调控。培养野生株和pkaR基因缺失突变株的萌发孢子,选择双氧水作为氧化压力与萌发孢子孵育。萌发孢子经过氧化压力刺激后,通过亚甲基蓝染色和再生实验检测经氧化压力刺激后萌发孢子活性;DCFH-DA染色检测在相同氧化压力条件下野生株和突变株萌发孢子内的ROS产生情况;TUNEL实验检测萌发孢子的DNA断裂情况,作为判断萌发孢子凋亡情况的依据;PI染色检测经氧化压力作用后萌发孢子细胞膜的完整性。结果:亚甲基蓝实验结果显示,经氧化压力处理pkaR基因缺失突变株萌发孢子的存活率明显高于野生株;胞内ROS含量检测结果显示,较低强度的氧化压力条件下WT株萌发孢子的ROS产生被促进,较高强度的氧化压力条件下ROS产生被抑制,与野生株相反突变株在较低氧化压力条件下ROS产量低,较高氧化压力条件下ROS产量高,在氧化压力条件下野生株和突变株萌发孢子的ROS含量有显著差异;TUNEL实验表明,随着氧化压力的提高,野生株凋亡的萌发孢子数量经历升高再降低的过程,而突变株凋亡的萌发孢子数量无显著变化,野生株与突变株相比凋亡的萌发孢子数量有显著差异;PI实验表明,在氧化压力条件下WT株的萌发孢子细胞膜受到的损伤更大,即坏死细胞更多,与突变株相比对于氧化损伤的抵抗能力更弱。结论:亚甲基蓝染色作为一种检测菌体存活率的方法,可以应用于烟曲霉萌发孢子活性的检测中;pkaR基因敲除后突变株的萌发孢子存活率更高,对于氧化压力的敏感性降低,说明pkaR基因影响烟曲霉萌发孢子对于氧化损伤的抵抗;经氧化压力刺激后,野生株与突变株的ROS产量有显著差异,说明pkaR基因参与调节烟曲霉萌发孢子ROS产生;在相同氧化压力条件下,野生株与突变株凋亡的萌发孢子数量有显著差异,说明pkaR基因参与调控烟曲霉萌发孢子的凋亡。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R379

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