【作者】 王丽欣；

【导师】 陈官芝；

【作者基本信息】 青岛大学 ， 皮肤病与性病学， 2010， 硕士

【摘要】 目的：通过对白念珠菌酵母状态和芽管状态,以及浮游状态和生物膜状态下ALS4、ALS9基因mRNA表达水平的比较,探讨这两种基因在白念珠菌表型转换及生物膜形成中可能发挥的作用,为进一步研究白念珠菌的发病机制提供理论依据。方法：选取3株标准株白念珠菌和10株临床株白念珠菌进行实验研究。白念珠菌酵母状态的培育和芽管状态的诱导以及浮游状态和生物膜状态的制备：GBS培养基,25℃振荡培养48h,收获酵母状态；pH7.0的RPMI-1640液体培养基,37℃震荡培养3h,收获芽管状态；体外6孔细胞培养板涂布小牛血清,RPMI-1640液体培养基,37℃静置培养48h,收获生物膜状态。液氮反复冻融加Trizol法提取不同生长状态白念珠菌的总RNA, RNA的纯度经紫外分光光度计A260/280比值估计,RT-PCR两步法扩增ALS4、ALS9基因mRNA,及内对照ACT1基因mRNA,扩增产物琼脂糖凝胶电泳,凝胶成像系统拍照。采用QuantityOne电泳分析软件对电泳条带进行分析,应用SPSS医学统计软件包两样本均数配对资料的t检验对实验数据进行统计学处理(以P<0.05为显著性标准)。结果：标准株白念珠菌芽管状态的ALS4基因mRNA(P<0.05)和ALS9基因mRNA(P<0.05)相对表达水平均明显高于酵母状态,临床株白念珠菌芽管状态的ALS4基因mRNA(P<0.05)和ALS9基因mRNA(P<0.05)相对表达水平均明显高于酵母状态；标准株白念珠菌生物膜状态的ALS4基因mRNA(P<0.05)和ALS9基因mRNA(P<0.05)相对表达水平均高于浮游状态,临床株白念珠菌生物膜状态的ALS4基因mRNA(P<0.05)和ALS9基因mRNA(P<0.05)相对表达水平均高于浮游状态。结论：ALS4基因和ALS9基因可能在白念珠菌的酵母相向菌丝相转换和体外生物膜形成中起重要作用。更多还原

【Abstract】 Objective To evaluate the role of Candida albicans ALS gene in transformation and biofilm formation, to provide theoretical basis for Candida albicans pathogenesis so that compare the expression of ALS gene mRNA level of Candida albicans between yeast and hyphal, between plantonic and biofilm.Methods Three C.albicans standard and ten clinical strain were induced to form yeast form in the condition cultured in GBS culture medium for 48h at 25℃, at planktonic condition. Germ tubes were induced by cultured in RPMI-1640 medium for 3h at 37℃, pH value at 7.0 with the shake speed of 150rpm. Biofilm models in vitro were formed on 6-well cell culture plate in RPMI-1640 media., after 48h incubation, the biofilm-grown cells were harvest. Liquid nitrogen and Trizol method were performed to isolate total RNA from the four different biological status separately. The purity of RNA was measured by absorption at 260/280nm with spectrophotometer. A semi-quantitative RT-PCR two-step method was developed to determine ALS4 and ALS9 genes against the housekeeping gene ACT 1.The identity of cloned fragments was confirmed with agarose gel electrophoresis. Gene expression leves were assayed by gel documentation system and Quantity One software. The paired test was used to analyze the experimental data.Result The RT-PCR results showed that the expression levels of ALS4 mRNA (P <0.05) and ALS9mRNA (P<0.05) of the standard strains were higher in germ tube compared with yeast form. Expression levels of ALS4 mRNA (P<0.05) and ALS9 mRNA (P<0.05) of the clinical strains were higher in germ tube compared with yeast form. Expression levels of ALS4 mRNA (P<0.05) and ALS9mRNA (P<0.05) of the standard strains in biofilm increased compare with planktonic cells.There were also increases in biofilm cells compared with the planktonic cells in the expression levels of ALS4mRNA(P<0.05) and ALS9mRNA (P<0.05) of the clinical strain.Conclusion The ALS4 and ALS9 gene may play an important role in Candida albicans transformation from yeast to germ tube and in biofilm formation in vitro.更多还原