【作者】 刘洪涛；

【导师】 姜远英；

【作者基本信息】 第二军医大学 ， 药理学， 2002， 硕士

【摘要】 羊毛甾醇14α-去甲基化酶(CYP51) 是真菌细胞膜必需成分-麦角甾醇生物合成过程中的一个关键酶。氟康唑、伊曲康唑、酮康唑等目前临床最常用的唑类抗真菌药物都是通过抑制这个靶酶来发挥抗真菌作用的。研究发现CYP51的结构变异能导致真菌耐药性的产生，因此研究CYP51的结构和功能关系是指导真菌耐药机制研究和抗真菌药研发的有效途径之一。 ．研究CYP51变异与白念珠菌耐药的关系，可采取两种策略：① CYP51定点突变：根据对CYP51活性中心的研究，选取反应活性中心的氨基酸残基，用重组PCR技术将其突变，考察突变型CYP5l的活性和功能上的变化。② 比较耐药菌株和敏感菌株CYP5l活性差异：采用敏感菌株、临床耐药菌株和氟康唑诱导的实验室耐药菌株，比较其CYP51活性和对氟康唑敏感性的差异，而后再研究其CYP5l的变异情况。本文在第一部分中利用体外氟康唑诱导获得了耐药白念珠菌，并采用微量液基稀释药敏实验法，测定了氟康唑对几株白念珠菌的最小抑菌浓度(MIC80) ，包括敏感菌株和临床耐药株、实验室耐药株，并检测了其对不同药物的交叉耐药性。敏感白念珠菌在含氟康唑的培养液中传代培养60天就会获得高度而耐药性，其MIC80可达到≥128μ／ml(敏感菌株MIC值一般<4μg／m1) 。利用该实验室诱导的耐药白念珠菌，研究其CYP51酶活性，并与亲本敏感株相比较。第二部分中我们建立了气一质联用测定法(GC-MS)，并用GC-MS法对敏感和耐药的白念珠菌细胞膜甾醇类物质(CYP51酶的底物和产物)的含量进行了检测。敏感株在无氟康唑的情况下，细胞膜的麦角甾醇成分含量占总甾醇类物质的60％以上，而随着氟康唑浓度的增大，CYP51酶受抑制，麦角甾醇成分的降低和羊毛甾醇等成分的增加与氟康唑呈剂量依赖性关系。在白念珠菌麦角甾醇生物合成通路中，Trimethyl(24-methylenelanost-8-en-3-o1) 即24(28) -亚甲基-24，25-二氢羊毛甾醇是CYP51酶的直接反应底物，随氟康唑剂量增加，其在胞膜中的累积增加量最为明显。而对氟康哗耐药的菌株，细胞膜中踊醇类的组成与敏感株差异显著，其中y01． 09▲中的麦角甾醇含量仅为30％左右；氟康唑作用后，细胞膜中的各甾醇成分含量的变化与敏感菌株相比，也有显著的二 差异，提示真菌细胞膜中涮醇成分的变化与其耐药性的形成有一定的关系。 为进一步定量研究耐药和敏感真菌CYP51酶的活性差异，我们又利用［l－“C」 乙酸整体细胞掺入实验和离细胞酶「2－乙甲羟戊酸掺入实验分别测定了氟康 哗对不同菌株胞膜中麦角吊醇生物合成的半数抑制浓度u0％inhibitLn of incorporation，IC扣人在真菌整体细胞的卜 口乙酸掺入实验中，随着氟康哗 浓度的提高，敏感菌胞膜中麦角由醇含量减少与羊毛洗醇及其上游成分含量的 累积呈明显的剂量依赖性，氟康哗对 yol．09麦角沿醇生物合成的r。仅为 16．0旭加，说明敏感白念珠菌的CYP51酶对氟康哗的敏感性高。而对于其它 几株耐药的白念珠菌，在相同实验条件下，氟康哗对其作用减弱，其IC扣值有 不同程度的提高，其中对010213＼ IC50达到了 313．3 og／of，与亲本敏感株 相比提高显著。在离细胞酶「2－’川甲羟戊酸掺入实验中，氟康哗对敏感株 y01．09麦角涮醇生物合成的IC50为19．709／01，对实验室耐药株yol．09‘的IC；。 为28．sng／ml，两者有显著差异，对其它耐药株的IC。。增加更为明显，其中对 010213‘的 IC；。则达至了 183．0 ny／＊1。提示 y01．09‘和其它耐药株的 CYP51酶 活性有明显差异。 本课题的目的是建立研究 CYP51对麦角沿醇生物合成活性的可靠方法，为 研究定点突变及耐药菌中CYP51的活性变化奠定基础。上述研究为真菌CYP51 酶的结构和功能研究奠定了基础。在后续的实验中，我们将进一步对耐药菌的 CYPSI基回进行测序比较，为研究真菌耐药机制，寻找广谱、高效、低毒的抗 真菌新药奠定基础。更多还原

【Abstract】 The cytochrome P450 14ot-lanosterol demethylase (CYP51) of fungi is involved in an important step in the biosynthesis of ergosterol which is essential for fungi to build their plasma membrane and is the target enzyme of azole drugs (fluconazole, ketoconazole and itraconazole, etc.), but it is prone to alterations leading to resistance to these agents. Azole resistance in the pathogenic yeast Candida albicans is an emerging problem in the HIV-infected population.We have been interested in understanding the molecular mechanisms of azole resistance in Candida albicans , so it is important to study the structure and function of CYPSl.Two strategies were adopted to research for it: Site-directed mutagenesis was performed to estimate effect of each of those mutations on resistance to azole derivatives.T315A, R467K, G464S mutant CYP51 have been researched by this mothod. (2) To find the mutagenesis from resistant clinical isolates or from induced-resistant strains in vitro.In this study, several Candida albicans isolates (include azole-resistant and susceptible strains) were tested. We obtained fluconazole-resistant strains(MIC 128ug/ml) induced by serial subcultures in YEPD containing different concentration of fluconazole. Sterol isolation and identification were analyzed by gas chromatography-mass spectrometry (GC-MS) to determine the cause for the drug tolerance. When grown in the absence of fluconazole, two sensitive strains and one resistant strain were found to contain ergosterol as their predominant sterol.In contrast, the ergosterol levels in two resistant isolates were comparatively low. When exposure to fluconazole, the decrease in the ergosterol level and the rise in the trimethyl(24-methylenelanost-8-en-3-ol) level were dosage-dependence, but there were many differences between azole-resistant and susceptible strains. It suggested the CYP51 of resistant isolates was less sensitive to fluconazole.The inhibitory effect of fluconazole on ergosterol biosynthesis in whole-cell fungi was measured by the incorporation of [l-14C]acetate into nonsaponifiable5lipids(NSLs). Compared to sensitive isolates, the half inhibitory concentration(IC5o)of fluconazole for ergosterol rise obviously in resistant isolates. IC5o to 010213A was 313.3 ng/ml. While the effect in cell-free extracts was measured by the incorporation of [2-’4C]mevalonate into NSLs. Compared to azole-susceptible strains, ICjo to CYP51 rised significant in azole-resistant strains. y01.09A from 28.5 ng/ml to 40.0 ng/ml, but others rised more significant .There were differences between azole- resistant strains. It suggested that the CYP51 of y01 .09A distinct from other azole-resistant strains.The aim of this topic is to establish a reliable method for the research of CYP51 on biosynthesis of ergosterol, settle foundation for the research of mutagenesis in resistant fungi and the structure and function of CYP51. In successive experiments, we will sequence and compare the CYP51 genes of the resistant isolates to inspect the molecular mechanisms of azole resistance in Candida albicans.更多还原