【作者】 杨敏；

【导师】 张宏；

【作者基本信息】 暨南大学 ， 皮肤病与性病学， 2003， 硕士

【摘要】 目的： 验证已知的白念珠菌菌丝相细胞的理想培养条件；通过对氟康唑敏感程度不同的白念珠菌酵母相和菌丝相细胞氟康唑作用靶酶基因ERG11序列的比较研究，以探讨两种细胞形态在基因序列上的差异性，从而为白念珠菌的病原真菌学研究其实验对象的选择(菌丝相或酵母相细胞)提供理论依据。 方法： 以同一母体来源的16株对氟康唑敏感程度不同的白念珠菌作为研究对象(分别为CA-1，CA-2，…，CA-17，无CA-10)，采用RPMI1640培养基(含10％的小牛血清)，37℃，连续传代培养7天，共转种12次，记录不同观察时间菌丝相和酵母相细胞数，计算菌丝相细胞形成率。分别提取酵母相和菌丝相细胞的基因组DNA。设计7对引物，分段扩增白念珠菌酵母相和菌丝相细胞的ERG11基因。扩增产物单链变性后，进行非变性聚丙烯酰胺凝胶电泳(PCR-SSCP)，观察结果并选择两种细胞形态间具有差异的片段行DNA序列测定。 结果： 1．以RPMI1640(pH=7.5，含10％小牛血清)为培养基，37℃传代培养7天(转种12次)后，每株白念珠菌的菌丝相细胞形成率均达99％以上； 2．ERG11的分段扩增结果显示每株白念珠菌均能扩增出目的片段，不存在敏感菌株和耐药菌株的差异； 3．SSCP分析及DNA序列测定结果显示白念珠菌的酵母相和菌丝相细胞ERG11基因序列存在多位点的差异。 结论： 1．RPMI1640(pH=7.5，含10％小牛血清)，37℃传代培养7天(转种12次)是获得白念珠菌菌丝相细胞的理想条件； 2．白念珠菌的酵母相和菌丝相细胞间部分基因序列存在差异； 3．白念珠菌的菌丝相细胞是进行其病原真菌学研究的适宜研究对象。更多还原

【Abstract】 Objective:To confirm the suitable hyphal form inducing medium which we have known. To analyze thedifferences on gene sequences between yeast form and hyphal form of Candia albicans, ERG 11gene encoding the target enzyme of fluconazole and its sequences were compared in this study. The evidence for selecting the form of Candiada albicans in medical mycology would be provided.Methods:A series of 16 strains Candida albicans from the same body (CA-1, CA-2, …A-17, but without CA-10 ) were induced to form hyphae or/and pseudo-hyphae in RPMI1640 medium with 10% calf serum at 37癈 for seven days bytransferring twelve times. The yeast form and hyphal form cells were counted and theI ratio of hyphal form cells was caculated. Seven sets of primers were designed toamplify the ERG 11 gene. DNA sequences of these fragments were compared through Single-strand conformation polymorphism (SSCP) analysis and DNA sepquencing.Results:1. The ratio of hyphal form cells of all the strains was more than 99% afer beingcultured for seven days by transferring twelve times in RPMI1640 medium with 10% calf serum at 37℃.2. The ERG 11 gene of all strains had been amplified triumphantly.3. There were some differences on ERG11 DNA sequences between yeast forrh and hyphal form of Candida albicans.Conclusion:1. RPMI1640 medium with 10% calf serum to induce hyphal form at 37℃ forseven days was thought to be the favorable condition for Candida albicans hyphal form indution.2. There were some differences on gene sequences between yeast form and hyphal form of Candida albicans.3. Hyphal form of Candida albicans was thought to be the appropriate object of medical mycology.更多还原