白色念珠菌磷脂酶与其毒力因子、耐药性及基因多态性的关系研究

发布时间：2018-01-04 09:35

本文关键词：白色念珠菌磷脂酶与其毒力因子、耐药性及基因多态性的关系研究　出处：《兰州大学》2010年硕士论文　论文类型：学位论文

【摘要】： 背景与目的白色念珠菌的毒力因子主要包括：细胞表面粘附结构、利于侵入的酶(天冬氨酸蛋白酶、磷脂酶)、表型转换等。宿主细胞膜中的主要成分磷脂,可被白色念珠菌分泌的磷脂酶所分解,白色念珠菌通过完整性受损的细胞膜侵入宿主细胞。目前,还不清楚磷脂酶和白色念珠菌其他毒力因子之间存在怎样的关系,因此,本实验检测临床分离的白色念珠菌磷脂酶活性,将其分为磷脂酶高活性组和磷脂酶低活性组,探讨磷脂酶与菌株其他毒力因子(溶血活性、细胞表面疏水性、粘附颊粘膜上皮细胞)、耐药性及基因多态性之间的关系,从而对深入了解磷脂酶在白色念珠菌致病性和耐药性中的作用提供实验依据,为抗真菌药物的研制及白色念珠菌的诊断和治疗奠定理论基础。方法采用蛋黄平板法对80株白色念珠菌的磷脂酶活性进行检测,以PZ值表示磷脂酶活性,根据菌株PZ值,选出磷脂酶活性较高和磷脂酶活性较低的菌株各10株,分为两组。比较两组的溶血活性、细胞表面疏水性、粘附颊粘膜上皮细胞能力的差异,分析白色念珠菌磷脂酶与上述三种毒力因子之间的关联性；同时进行药物敏感性检测,比较两组间耐药性的差异；提取菌株DNA,应用随机扩增多态性DNA(RAPD)技术分析两组的基因型特征。结果从临床分离的80株白色念珠菌中,78株可以分泌磷脂酶,磷脂酶阳性检出率为97.5%,其PZ值的范围为0.566～1.000,A组PZ均值为0.639±0.043,B组PZ均值为0.847±0.079,两组有显著性差异(p0.05)。两组的溶血活性、细胞表面疏水性均无显著性差异(p0.05),菌株磷脂酶活性与溶血活性、细胞表面疏水性均无直接关联(r=0.122,p0.05)、(r=0.072,p0.05)。而两组的粘附力有显著性差异(p0.05),A组菌株对颊粘膜上皮细胞的粘附力显著强于B组,磷脂酶活性与粘附力呈正相关(r=0.773,p0.01)。两组菌株对7种抗真菌药物的耐药性明显不同,A组中对益康唑耐药率为70%,对咪康唑、氟康唑、伊曲康唑也有中敏或者耐药情况；而B组中对益康唑耐药率仅为10%,对其余的6种药物均敏感。RAPD结果显示,除B组中两株磷脂酶阴性的菌株显示为独特的基因型外,剩余A、B两组菌株主要条带的数量和位置差异较小,在1300 bp处均显示一高亮度条带,在1500 bp处各显示有3条亮带,遗传相似系数在0.500～1.000之间,两组菌株基因多态性无明显差异。结论临床分离的白色念珠菌多数可以产生磷脂酶,说明磷脂酶是白色念珠菌重要的毒力因子；其活性与菌株的粘附力有一定的相关性,但与菌株的溶血活性、细胞表面疏水性并没有直接的相关,磷脂酶活性不能直接反应菌株其他毒力因子的情况；磷脂酶活性高的菌株其耐药性也随之增强,表明磷脂酶和耐药有一定的相关性；基因多态性不能直接反应磷脂酶活性的强弱,说明基因多态性是对包括溶血活性、细胞表面疏水性、粘附力及耐药等特征的一个综合反映,不只简单地反映磷脂酶这一单一的毒力特征。
[Abstract]:Background and objective virulence factors of Candida albicans include: cell adhesion structure, to invade the enzyme (aspartic proteinase, phospholipase), phenotypic conversion. The main component of host cell membrane phospholipids, can be decomposed by Candida albicans Candida albicans secreted phospholipase, through the cell membrane integrity damage of host cell invasion at present, is not clear what relationship exists between, phospholipase of Candida albicans and other virulence factors. Therefore, the detection of clinical isolates of Candida albicans phospholipase activity, which can be divided into high activity of lipase and phospholipase P group low activity group, and other virulence factors of phospholipase strain (hemolytic activity, cell surface hydrophobicity, adhesion buccal mucosa epithelial cells), the relationship between the drug resistance and the gene polymorphism, and in-depth understanding of phospholipases in pathogenicity and drug resistance in Candida albicans The effect provides an experimental basis for the development of antifungal drugs and the theoretical basis for the diagnosis and treatment of Candida albicans.
Methods using egg plate assay of phospholipase activity of 80 strains of Candida albicans, with a value of PZ indicates the phospholipase activity, according to the strains of PZ, selected high phospholipase activity and phospholipase activity in low strain all 10 strains were divided into two groups. The hemolytic activity of two groups, cell surface hydrophobicity, adhesion ability between buccal mucosal epithelial cells, analyzing the relationship between Candida phospholipase and above-mentioned three kinds of virulence factors; and drug sensitivity test, were compared between the two groups of drug resistance strain DNA; extraction, using random amplified polymorphic DNA (RAPD) technology analysis of genotype characteristics of the two groups.
The results from 80 clinical isolates of Candida albicans, 78 strains secreted phospholipase, phospholipase a positive rate of 97.5%. The PZ value range is 0.566 ~ 1 A, PZ value was 0.639 + 0.043, B + 0.079 PZ value was 0.847, there was significant difference between two groups (P0.05). The hemolytic activity the two group, there were no significant differences in cell surface hydrophobicity (P0.05) strains, and the hemolytic activity of phospholipase activity, cell surface hydrophobicity had no direct correlation (r=0.122, P0.05), (r=0.072, P0.05). There was significant difference between the two groups of adhesion, adhesion (P0.05) strains of buccal mucosa the epithelial cells of A group were significantly higher than that in group B, positive correlation of phospholipase activity and adhesion (r=0.773, P0.01). Two groups of drug resistance strains to 7 kinds of antifungal drugs were significantly different, A group of econazole drug resistance rate was 70%, fluconazole and miconazole, I Mako, also has the sensitivity of or resistance; B group To Econazole resistance rate was only 10%, were sensitive to 6 drugs for the rest of the.RAPD results showed that in B group in two strains of negative strains showed phospholipase a unique genotype, residual A, B two groups were mainly a number and position difference with smaller, at 1300 BP showed a high brightness bands at 1500 BP each showed 3 bright bands. The genetic similarity coefficient among 0.500 ~ 1, no significant difference between the two groups was gene polymorphism.
Conclusion most clinical isolates of C.albicans can produce phospholipase, phospholipase is an important virulence factor of Candida albicans; there is a certain correlation between the activity and adhesion of strains, but the hemolytic activity and strain, cell surface hydrophobicity is not directly related, phospholipase activity not a direct response to strains of other virulence factors; phospholipase activity high strain resistance also increases, indicating that there is some correlation between phospholipase and resistance; gene polymorphism is not a direct response to the intensity of phospholipase activity, indicating gene polymorphism is to include hemolytic activity, cell surface hydrophobicity, a comprehensive reflection of the adhesion and resistance characteristics, not simply reflect a single phospholipase the characteristics of virulence.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R379

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