舒洛地尔对白假丝酵母菌生物被膜形成及生长的作用研究

发布时间：2018-03-11 03:01

本文选题：白假丝酵母菌　切入点：生物被膜　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：白假丝酵母菌(Candida albicans)是一种常见的人类致病真菌,据疾病控制中心(CDC)统计结果显示,在口腔假丝酵母菌病发病率中,儿童的发病率为7%,艾滋病病人为31%,癌症化疗患者为20%;在健康女性中,假丝酵母菌阴道炎也十分常见,临床表现多呈反复发作。由于白假丝酵母菌可以通过形成生物被膜以应对多种复杂的环境,传统的抗真菌药物难以穿透从而导致白假丝酵母菌难以清除。白假丝酵母菌生物被膜的形成包括四个阶段:粘附期,定植初期,细胞外基质物质分泌期,成熟期。生物被膜是白假丝酵母菌重要的毒力因子之一,菌可借此粘附在宿主组织,假体,植入物(如导管,心脏瓣膜等)的表面,以利于白假丝酵母菌的定植。并且,生物被膜还能够显著增加白假丝酵母菌对传统抗真菌药物的耐药性,与浮游细胞相比,生物被膜形态的白假丝酵母菌对唑类药物的耐药性升高约1000倍。因此,亟待寻找新的抗真菌药物来应对白假丝酵母菌感染。舒洛地尔(1-(4-异丙基苯硫基)-2-氨基-辛基丙醇)是一种钙通道阻滞剂,能够抗血管痉挛、抗血栓形成。有研究发现,舒洛地尔在体外能够抑制白假丝酵母菌的生长;还有研究证实,舒洛地尔联合氟康唑用药,对新型隐球菌有抑制作用,而舒洛地尔对白假丝酵母菌生物被膜的作用,目前未见相关研究报道。本研究通过检测舒洛地尔对白假丝酵母菌生物被膜形成及成熟生物被膜的抑制作用来评估其对生物被膜相关的白假丝酵母菌病的治疗潜能。目的研究舒洛地尔对白假丝酵母菌生物被膜的抑制作用。通过体外实验评价舒洛地尔抑制生物被膜的效果及初步探究其抑制白假丝酵母菌生物被膜的作用机制;通过体内实验评价舒洛地尔对白假丝酵母菌阴道炎的治疗效果,以期为浅表性白假丝酵母菌生物被膜感染的治疗提供新策略。方法1.运用XTT减低法测定舒洛地尔对白假丝酵母菌YEM30,临床分离株LC3浮游细胞最小抑菌浓度(MIC_(80)),生物被膜抑制浓度(BIC_(80)),生物被膜根除浓度(BEC_(80))。2.通过时间杀菌曲线检测舒洛地尔抑制白假丝酵母菌生物被膜形成、成熟期生物被膜的动力学。3.运用扫描电子显微镜观察舒洛地尔对白假丝酵母菌生物被膜的形态影响。4.运用透射电子显微镜观察舒洛地尔对白假丝酵母菌生物被膜细胞超微结构的影响。5.运用倒置显微镜观察舒洛地尔在不同加药时间对白假丝酵母菌生物被膜酵母相-菌丝相形态转变影响。6.运用qRT-PCR技术检测舒洛地尔对白假丝酵母菌菌丝生长特异性相关基因的表达影响。7.构建白假丝酵母菌阴道炎模型,评价舒洛地尔在体内抗白假丝酵母菌生物被膜效果。结果1.舒洛地尔可抑制白假丝酵母菌生物被膜的形成,并可破坏成熟的生物被膜。舒洛地尔对白假丝酵母菌YEM30,临床分离株LC3 MIC_(80)为4 μg/mL,BIC_(80)为16 μg/mL,BEC_(80) 为 64 μg/mL。2.舒洛地尔抑制白假丝酵母菌生物被膜细胞的代谢活性呈浓度依赖性,随着作用时间延长,浓度越高,抑制效果越好。3.舒洛地尔在体外使白假丝酵母菌生物被膜细胞皱缩,随着作用时间延长,出芽细胞脱落,皱缩明显。4.舒洛地尔在体外能够破坏白假丝酵母菌生物被膜细胞的超微结构,使细胞膜皱缩,细胞壁断裂,细胞内电子密度降低,细胞内容物渗出。5.舒洛地尔有效阻止白假丝酵母菌从酵母相向菌丝相转变,舒洛地尔作用时间点越早,作用越明显。6.舒洛地尔在体外使白假丝酵母菌菌丝生长特异性相关基因HWP1,ALS3,ECE1的表达下调。7.动物实验显示,与对照组相比,用药组的菌落数显著减少,炎症减轻,舒洛地尔能有效治疗白假丝酵母菌阴道炎。结论研究结果显示,舒洛地尔在体内外均能有效抑制白假丝酵母菌生物被膜。体外实验表明舒洛地尔不仅能破坏成熟生物被膜的形态和超微结构,而且通过下调菌丝特异性相关基因HWP1,ALS3,ECE1的表达,阻断菌丝相表达,从而抑制白假丝酵母菌生物被膜的形成。白假丝酵母菌阴道炎动物模型的研究表明,用药组的菌落数显著减少,炎症减轻。综上所述,我们的研究表明舒洛地尔对于浅表性白假丝酵母菌生物被膜感染具有较理想的抑制作用,是一种潜在的、有价值的治疗手段。
[Abstract]:Candida albicans (Candida albicans) is a common human pathogen, according to the Centers for Disease Control (CDC) statistics show that in the oral candidiasis incidence in children, the incidence of 7% AIDS patients, 31% patients with cancer chemotherapy was 20%; in healthy women, Candida yeast vaginitis is also very common, the clinical manifestations were recurrent. Due to Candida albicans can form biofilms in response to a variety of complex environment, the traditional antifungal drugs to penetrate causing Candida albicans is difficult to remove. Candida albicans biofilm formation includes four stages: adhesion. Early colonization, extracellular matrix material secretion period, mature period. The biofilm is one of the important virulence factors of Candida albicans, bacteria can take the adhesion in the host tissue, prosthesis, implants (such as catheters, heart valve and so on) the table The surface, in order to Candida albicans colonization and biofilm also significantly increases the resistance of Candida albicans to traditional antifungal drugs, compared with planktonic cells, drug resistance of Candida albicans by biological membrane morphology of azoles increased about 1000 times. Therefore, it is urgent to find to deal with white Candida infection of new antifungal agents. Suloctidil (1- (4- isopropyl phenylthio) -2- amino - octyl alcohol) is a calcium channel blocker, can anti vasospasm, thrombosis. Studies have found that suloctidil could inhibit Candida albicans in vitro and growth; study confirmed that suloctidil fluconazole treatment has inhibitory effect on Cryptococcus neoformans and suloctidil Candida albicans biofilm, currently no reports about this research. By detecting suloctidil Candida albicans Biological inhibition of membrane formation and mature biofilm to assess its effect on Candida albicans disease the therapeutic potential of biofilm related. Objective to study the inhibitory effect of suloctidil on Candida albicans biofilm. In vitro evaluation of suloctidil biofilm effect and explore its inhibition the mechanism of Candida albicans biofilm; in vivo evaluation of suloctidil Candida albicans vaginitis treatment, superficial provides a new strategy for the treatment of Candida albicans biofilms in order for infection. Methods 1. using XTT reduction method for the determination of suloctidil Candida albicans YEM30. Strain LC3 of planktonic cells minimal inhibitory concentration of clinical isolates (MIC_ (80)), biofilm inhibitory concentration (BIC_ (80)), biofilm eradication concentration (BEC_ (80).2.) by time killing curve detection to suppress white Luodier Shu Candida albicans biofilm formation, mature biofilm kinetics of.3. by scanning electron microscopy observation suloctidil on Candida albicans biofilm morphology by transmission electron microscope to observe the effect of.4. suloctidil on Candida albicans biofilm cell ultra microstructure effects by.5. inverted microscope suloctidil at different dosing time on Candida albicans biofilm - yeast phase of mycelial phase transformation effect of.6. using qRT-PCR technology to detect suloctidil Candida albicans mycelial growth of specific gene expression effect of.7. construction of Candida albicans vaginitis model, evaluate the suloctidil in vivo anti Candida albicans biofilm effect results 1.. Suloctidil can inhibit the formation of Candida albicans biofilms, and can damage the mature biofilm suloctidil with Candida. Albicans clinical isolates of LC3 YEM30, MIC_ (80) 4 g/mL, BIC_ (80) 16 g/mL, BEC_ (80) 64 g/mL.2. suloctidil inhibit Candida albicans biofilm metabolic activity of mesangial cells in a concentration dependent manner, with the extension of the time, the higher the concentration, inhibition the better.3. suloctidil in vitro to Candida albicans biofilm cell shrinkage, with the prolongation of time, budding cells shedding, obvious shrinkage.4. suloctidil can destroy the Candida albicans biofilm ultrastructure of cell membrane in vitro, the cell membrane shrinkage, cell wall fracture, the decrease of intracellular electron density.5., cell exudant suloctidil effectively prevent Candida albicans from yeast to hyphal phase transition, suloctidil time points earlier and more obvious effect of.6. suloctidil in vitro to Candida albicans hyphal growth specific genes related to H WP1, ALS3, ECE1 expression of.7. in animal experiments, compared with the control group, treatment group was significantly reduced the number of colonies, inflammation, suloctidil can be effective in the treatment of Candida vaginitis. Conclusion the results of the study showed that suloctidil could effectively inhibit Candida albicans biofilm in vitro and in vivo. In vitro experiments indicated that suloctidil can not only destroy the mature biofilm morphology and ultrastructure of the membrane, and through down-regulation of hypha specific genes HWP1, ALS3, ECE1 expression, blocking hyphal expression, thereby inhibiting the formation of Candida albicans biofilms. Research on Candida albicans vaginitis animal model showed that colony the number of treatment group was significantly reduced, reduced inflammation. In summary, our study shows that suloctidil for superficial Candida albicans biofilm can inhibit ideal membrane infection, is a kind of potential, Valuable treatment.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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