【作者】 苏惠春；

【导师】 程波； 张声；

【作者基本信息】 福建医科大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 目的：探讨白念珠菌双形态性形成的相关影响因素，寻找控制菌丝形成的关键基因。方法：1、分离培养八株不同来源的白念珠菌(标准菌株和临床标本分离菌株)，控制白念珠菌菌丝生长的影响因素，包括孢子浓度、培养温度、培养基成分和pH值，观察菌丝的生长特点和形成率的变化。2、分别采用-80℃研磨、液氮研磨、酸洗玻璃珠三种破壁方法破壁，用TRIzol试剂提取白念珠菌菌丝相和酵母相的总RNA，对提取的RNA进行定性定量及完整性分析。3、通过设置内参的半定量逆转录聚合酶链反应(RT-PCR)的方法测定白念珠菌双形态性的四个相关调控基因EFG1、HWP1、HYR1、HGC1的mRNA在菌丝相和酵母相中的表达差异。4、将八株白念珠菌分别接种在显色培养基和玉米吐温培养基上，用NB-UVB临床照射剂量(0.4-3.0J／cm~2)对其进行3次／天，共7天的照射，观察菌丝和酵母的生长及形态变化。结果：1、菌丝具有聚集生长成絮状物的特点。在pH=5.0时，菌丝上的孢子易脱落；在pH=9.0时，易形成较大分生孢子；中性pH值最有利于纯菌丝的生长。孢子浓度5×10~6／ml，在含10％胎牛血清pH7.0的PRMI1640培养基中，38℃静置培养4到8小时，菌丝的形成效果好。菌丝纯度大于95％。2、三种破壁方法提取白念珠菌酵母相RNA的浓度(x±s，ug／ul)分别为：-80℃研磨法(0.464±0.101，ug／ul)、酸洗玻璃珠法(0.911±0.413，ug／ul)、液氮研磨法(1.418±0.524，ug／ul)。而且后两者RNA的完整性比前者好。3、HWP1和HGC1基因在菌丝相有表达，而在酵母相无表达。EFG1和HYR1基因在菌丝相和酵母相的表达没有明显差异。4、随着UVB照射剂量的增加，菌丝形成逐渐减少，菌丝上生长的分生孢子逐渐增多。照射剂量超过1.5J／cm~2念珠菌的生长受到明显抑制。结论：1、血清、中性pH值、适当温度有利于菌丝生长。2、液氮研磨破壁和酸洗玻璃珠振荡破壁为较好的破壁提取白念珠菌RNA的方法，其中液氮研磨法最好。3、NB-UVB照射剂量在0.4—3.0J／cm~2的时候，可抑制菌丝生长。4、HWP1和HGC1基因为菌丝特异表达基因。更多还原

【Abstract】 Objective:To research some interfering factors act on dimorphism of Candida albicans. To find key genes which control hyphal formation of Candida albicans.Methods:1、Eight strains of Candida albicans culturing from different resoures (a standard strain and strains from clinical specimens) were tested. Interfering factors, such as spores concentration, cultural temperature, media components and pH were acted on the Candida albicans. The changes of hyphal formation ratio and growth features of Candida albicans were observed.2、Three breaking fungi cell wall methods were used in the experiments: Grinded Candida albicans in -80℃or in liquid nitrogen, and acid washed glass beads. Total RNA of hyphae and yeasts were extracted by RTIzol agent, and then analyzed the quality and integrity of extracted RNA.3、Analyzed mRNA different expressions of four genes(EFG1, HWP1, HYR1 and HGC1) which related to dimophism of Candida albicans by semiquantitative reverse transcriptase polymerase reaction with an intrinsic control.4、Exposed eight strains of Candida albicans to NB-UVB in clinical dosages three times daily for 7 days. Morphological changes and hyphal formation of the fungi were observed.Results:1、Hyphae became floccus in liquid media. Conidiosporespores failed easily at pH 5.0. Big conidiospore formed at pH9.0.Neutral pH is best for hyphaeformation. Hyphae developed well in RPMI1640 medium with 10 percents of fetal bovine serum, pH at 7.0, spore concentration was 5×10~6/ml, incubated at 38℃for four to eight hours. 2、Total RNA concentrations ((?)±s, ug/ul) were extracted with different methods were 0.464±0.101 ( grinded in -80℃), 0.911±0.413( acid washed glass beads ) and 1.418±0.524(grinded in liquid nitrogen ).The integrity of RNA with last two methods are better than the first one.3、HWP1 and HGC1 were expressed in hypha and negative in yeast of Candida albicans. EFG1 and HYR1 were expressed in both forms.4、Hyphae of candia albicans were reduced and blastspores were increased which paralleled with UVB irradiation dosage. Hyphae developing were inhibited obviously when irradiation dose was over 1.5 J/cm~2.Conclusions:1、Serum, neutral pH, adequate temparature are profitable to hyphal formation.2、Grinded candia albicans in liquid nitrogen and shocked candia albicans with acid washed glass beads are good broken-wall methods for RNA extration in Candida albicans. Among these three methods, Grinded in liquid nitrogen is the best.3、Hyphae are inhibited when the doses of UVB irradiation is between 0.4 J/cm~2 and 3.0J/cm~2.4、HWP1 and HGC1 are specific genes which control hyphal formation.更多还原