质膜蛋白3参与的真菌耐多烯类AmB机制研究

发布时间：2018-01-10 06:09

本文关键词：质膜蛋白3参与的真菌耐多烯类AmB机制研究　出处：《东华大学》2017年硕士论文　论文类型：学位论文

更多相关文章： AmB 耐药性 PMP3 ROS MAPK途径

【摘要】：深部真菌感染是一种致病性真菌侵犯皮下组织和器官所引起的真菌感染性疾病,其死亡率较高且难以治疗。两性霉素B(Amphotericin B,AmB)是一种有效治疗深部真菌感染的临床用药,但随着AmB使用的增多,耐药性问题日渐严峻,而且耐药菌株的分子机制并不清楚。本实验室前期研究表明质膜蛋白3(PMP3p)高表达增加了细胞对AmB的抗性,但PMP3与AmB相关性的研究还很少。本文开展质膜蛋白3在酿酒酵母(Saccharomyces cerevisiae)和白色念珠菌(Canidia Albicans)中参与的耐AmB机制研究。文献研究表明AmB的作用机制主要有两种:一种是通过与甾醇结合,使细胞质膜形成孔洞,胞内物质外流,造成细胞死亡;一种是AmB能增加细胞内的氧化损伤,造成细胞死亡。因此,首先,本文通过PI染色质完整性检测实验和罗丹明123吸收和外排实验,发现过表达PMP3能增加细胞质膜的稳定性。因PMP3,最早是在植物中发现的,且对植物细胞内离子的平衡有重要作用。因此我们探究了在酵母中,PMP3调控两性霉素B抗性与离子的关系。实验表明PMP3对Na+的平衡起重要作用,而与K+和Ca2+没有相关性。同时还发现,PMP3调节AmB的抗性与NHA1和VCX1通路是无关的。其次,我们在甾醇合成通路阻断的突变体(erg2△,erg6△,erg24△,hmg1△和hmg2△)中超表达PMP3基因也能够提高细胞对AmB的抗性,说明阻断甾醇合成通路并不影响PMP3调节细胞对AmB的抗性。进一步研究在野生型(BY4741)及不同遗传突变菌株中过表达PMP3甾醇含量没有显著变化。这表明PMP3不是通过调控甾醇的合成来影响细胞对AmB的抗性。最后,研究了PMP3与药物诱导的氧化胁迫之间的关系。结果表明AmB能造成细胞内活性氧的增加,使细胞内的脂质过氧化产物(TBRAS)和蛋白羰基含量(CP)增加。并且抗氧化相关的酶如超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(Gpx)活性也增加,超表达PMP3能使细胞内抗氧化酶合成升高,降低了细胞内的活性氧含量,从而保护了细胞,这表明超表达PMP3能提高细胞内抗氧化酶降低胞内活性氧来增加细胞对AmB的抗性。同时,已有研究表明细胞内ROS的降低不仅与抗氧化酶有关,而且与MAPK通路有密切的关系。我们进一步研究表明,MAPK通路的中的关键基因SPC1和ATF1缺失的情况下(atf1△和spc1△),超表达PMP3不表现AmB抗性,也不能降低细胞内活性氧;PT-PCR的检测细胞PMP3表达水平与AmB处理或氧化胁迫的依赖关系被破坏。总之,PMP3调节AmB的抗性依赖于MAPK通路的完整性。
[Abstract]:Deep fungal infection is a fungal infectious disease caused by pathogenic fungi invading subcutaneous tissues and organs, and its mortality is high and difficult to treat. Amb is an effective clinical drug for the treatment of deep fungal infections, but with the increasing use of AmB, the problem of drug resistance is becoming more and more serious. In addition, the molecular mechanism of drug-resistant strains is not clear. Our previous study showed that the high expression of plasma membrane protein 3PMP3p increased the cell resistance to AmB. However, the correlation between PMP3 and AmB was seldom studied. In this paper, the plasma membrane protein 3 was developed in Saccharomyces cerevisiae and Candida albicans (Candida albicans). Canidia Albicanss is involved in the mechanism of AmB tolerance. The literature shows that there are two main mechanisms of AmB: one is by binding with sterols. Make the cell plasma membrane to form a hole, intracellular material outflow, resulting in cell death; One is that AmB can increase the oxidative damage in cells and cause cell death. Therefore, firstly, the Pi chromatin integrity test and Rhodamine 123 absorption and efflux test were used. It was found that overexpression of PMP3 could increase the stability of cytoplasmic membrane. PMP3 was first found in plants and played an important role in the homeostasis of intracellular ions in plants. PMP3 regulates the relationship between amphotericin B resistance and ions. The results show that PMP3 plays an important role in Na balance, but has no correlation with K and Ca2. The resistance of PMP3 to AmB regulation was not related to the NHA1 and VCX1 pathway. Secondly, our mutant Erg2 / erg6 / Erg24 was blocked by sterol synthesis pathway. Overexpression of PMP3 gene in hmg1 and hmg2 can also enhance the cell resistance to AmB. It is suggested that blocking the sterol synthesis pathway does not affect the resistance of PMP3 to AmB. There was no significant change in the content of overexpressed PMP3 sterols in different genetic mutants, which indicated that PMP3 did not affect the resistance of cells to AmB by regulating the synthesis of sterols. The relationship between PMP3 and drug-induced oxidative stress was studied. The results showed that AmB could increase intracellular reactive oxygen species (Ros). The lipid peroxidation products (TBRAS) and protein carbonyl content (CPP) were increased, and antioxidant related enzymes such as superoxide dismutase (SOD), catalase (catalase). Glutathione peroxidase (GPX) activity was also increased. Overexpression of PMP3 could increase the synthesis of antioxidant enzymes and decrease the content of reactive oxygen species in the cells, thus protecting the cells. This suggests that overexpression of PMP3 can increase intracellular antioxidant enzymes and decrease intracellular reactive oxygen species (Ros) to increase cell resistance to AmB. Meanwhile, studies have shown that the decrease of intracellular ROS is not only related to antioxidant enzymes. Our further study shows that the key genes in the MAPK pathway SPC1 and ATF1 are absent in the presence of ATF1 and spc1). Overexpression of PMP3 did not show AmB resistance, nor did it reduce intracellular reactive oxygen species (Ros). The relationship between the PMP3 expression level of PT-PCR and AmB treatment or oxidative stress was destroyed. In a word, the regulation of AmB resistance by PMP3 depended on the integrity of MAPK pathway.
【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R379

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