【作者】 陈建宏；

【导师】 范瑞强；

【作者基本信息】 广州中医药大学 ， 中医外科学， 2009， 博士

【摘要】 目的随着临床治疗念珠菌性外阴阴道炎唑类药物的运用,其唑类药物耐药发生率也越来越高,氟康唑对于白念珠菌发生作用在于药物进入细胞内作用于相关基因或与一些关键酶结合而起到抑制作用;其耐药性的产生也是由于相关耐药基因表达或变异所致;患者病情缠绵难愈,已经引起广泛的重视;如何降低患者对哗类药物的耐药性,或者开发出治疗复发性念珠菌性外阴阴道炎的新方法、新药物,减轻患者痛苦,已是广大医药工作者面临的重大问题。研究发现,临床上中医中药治疗对于初发或是复发性念珠菌性外阴阴道炎效果良好,可以明显改善患者痛苦;根据以前的研究,中药香莲外洗液外洗治疗念珠菌性外阴阴道炎或复发性念珠菌性外阴阴道炎疗效显著,对阴道白念珠菌有明显的抑制作用。白念珠菌对唑类药物的耐药研究在现代分子生物学技术的指引下已经进入分子水平,并且在不断的发展中,国外现在已经定位到专门的耐药基因上,并对专门的耐药基因进行多方位的研究;国内的研究目前基本上着重于药物作用的基因的整体的研究,从整体上来阐述耐药的实质。本研究的创新点主要主要是借鉴现代分子生物学技术,在基因水平上揭示香莲外洗液对白念珠菌作用的分子机制,包括白念珠菌的敏感株和耐药株,揭示香莲外洗液的作用机理,分析不同菌株在外洗液作用下的基因表达差异,利用基因分析相关技术对中药外洗方作用的菌株进行多靶点超高通量筛选,构建香莲外洗液基因作用机制;中药治疗念珠菌病效果良好,但是其具体的作用机理是什么,同氟康唑一样有同样的作用机理,还是有自己的特异性未揭示的靶点;对于反复发作的念珠菌病,中药外用也有良效,那么对于耐药念珠菌株,其在分子作用水平上的机理又是什么呢?值得我们好好的研究,为中药制剂的规范化提供佐证;由此对中药的研究与开发及走向世界具有实际意义。方法1氟康唑诱导SC5314标准菌株为短期耐药株,观察诱导后和诱导前的基因差异表达。运用基因芯片对氟康唑诱导短期耐药的基因与原代菌株进行比较分析,在基因层面上了解耐药性的形成。2香莲外洗液作用于氟康唑诱导的SC5314短期耐药株,观察香莲外洗液作用前和作用后的相同差异基因表达。对香莲外洗液作用于氟康唑诱导的SC5314短期耐药株的基因变化与氟康唑诱导原代菌株至耐药菌株的基因表达变化进行比对,找出共同的差异表达基因,由此揭开香莲外洗液作用的特殊靶点并进行详细分析。3香莲外洗液作用于实验室诱导耐药菌株和临床耐药株后相同基因差异基因表达分析。对香莲外洗液作用的氟康唑诱导的短期耐药菌株和临床耐药菌株差异基因进行比对,找出共同的差异表达基因,由此揭开香莲外洗液作用的特殊靶点并进行详细分析。4对NCCLS的M27-A3、S3的MIC观测方法与葡萄糖消耗试验测量的MIC方法进行一致性研究。采用葡萄糖消耗试验测量香莲外洗液作用的菌株的MIC。结果1由氟康唑对三种菌株MIC测定结果可知,香莲外洗液作用于试验菌株SC5314其MIC为0.4883mg/ml。氟康唑作用于SC5314的MIC为0.25μg/ml,初步推断在体外抑菌白念珠菌SC5314试验中,香莲外洗液0.4883mg/ml和氟康唑0.25μg/ml效力相当。留待以后研究确证。2随着香莲外洗液的浓度上升,试验的两种菌株各孔葡萄糖浓度越高,说明葡萄糖消耗越少,经过药物浓度取对数后进行分析,发现取对数后的药物浓度和每孔中葡萄糖浓度者呈现线性关系;对香莲外洗液作用下的两种菌株的线性关系进行分析比较,发现香莲外洗液对两种菌株作用具有一致性。随着氟康唑药液的浓度上升,试验的两种菌株各孔葡萄糖浓度越高,说明葡萄糖消耗越少,经过药物浓度取对数后进行分析,发现取对数后的药物浓度和每孔中葡萄糖浓度者呈现线性关系;对氟康唑作用下的两种菌株的线性关系进行分析比较,发现对两种菌株作用具有不一致性。3氟康唑诱导的耐药SC5314菌株与诱导前的敏感株相比,其差异基因表达主要集中在:编码蛋白酶体及蛋白水解酶的基因,编码糖脂代谢过程中相关蛋白的基因,细胞周期相关基因,转录及翻译调节相关基因,应激反应相关基因,线粒体相关基因,细胞壁功能相关基因。4白念珠菌SC5314诱导耐药株经香莲外洗液作用后,其表达变化的基因主要是:编码应激反应家族蛋白的基因,生物膜相关性基因,编码蛋白酶体基因一族,编码细胞周期相关蛋白基因,转录及翻译调节的相关蛋白基因。5白念珠菌临床耐药菌株经香莲外洗液作用后,其表达变化的基因主要是:编码热休克蛋白基因,丝氨酸/苏氨酸蛋白激酶基因,蛋白酶体家族基因,调控转录及翻译基因。结论由氟康唑对三种菌株MIC测定结果可知,质量控制菌株ATCC22019和TCC6258经过两次重复试验,与生长对照孔进行比较;其结果均落在CLSI推荐的M27—S3 24小时标准范围内,从结果来看,质控菌株对氟康唑敏感性均在控,说明试验条件、操作等均符合要求。因此氟康唑对于SC5314的MIC为0.25μg/ml其结论是可信的。香莲外洗液和氟康唑不同,对于氟康唑,由于它是治疗念珠菌感染的常用的西药,在世界范围内有广泛的临床运用,对于念珠菌(酵母菌)MIC的测定在国际上有深入的研究,氟康唑作用于念珠菌的MIC经过系统的研究有一个系统的范围(标准)可以供我们参考。而香莲外洗液则不同,作为一种医院自制外用药剂,虽然其开发运用超过10,但是也仅是局限于本院使用,对于香莲外洗液作用于念珠菌的系统的MIC的研究尚未启动,因此也谈不上有香莲外洗液作用于念珠菌的MIC可供参照的标准。因此,试验对香莲外洗液作用于念珠菌的MIC测定也进行了初步的探索,为以后的研究打开思路。试验中,香莲外洗液作用于试验菌株SC5314其MIC为0.4883mg/ml。氟康唑作用于SC5314的MIC为0.25μg/ml,基本可以推断在体外抑菌白念珠菌SC5314试验中,香莲外洗液0.4883mg/ml和氟康唑0.25μg/ml效力相当。香莲外洗液和氟康唑对念珠菌葡萄糖消耗的影响与分析研究发现随着香莲外洗液的浓度上升,试验的两种菌株各孔葡萄糖浓度越高,说明葡萄糖消耗越少,经过药物浓度取对数后进行分析,发现取对数后的药物浓度和每孔中葡萄糖浓度者呈现线性关系;对香莲外洗液作用下的两种菌株的线性关系进行分析比较,发现香莲外洗液对两种菌株作用具有一致性。随着氟康唑药液的浓度上升,试验的两种菌株各孔葡萄糖浓度越高,说明葡萄糖消耗越少,经过药物浓度取对数后进行分析,发现取对数后的药物浓度和每孔中葡萄糖浓度者呈现线性关系;对氟康唑作用下的两种菌株的线性关系进行分析比较,发现对两种菌株作用具有不一致性。这可能说明两种药物的作用机制不一样,氟康唑主要作用于细胞色素P450固醇合成酶途径,而香莲外洗液作用于什么途径却不得而知;这也许就是香莲外洗液和氟康唑作用的不同之处,有待于以后进一步研究。对氟康唑诱导的耐药SC5314菌株与诱导前的敏感株进行基因芯片研究发现,其差异基因表达主要集中在:编码蛋白酶体及蛋白水解酶的基因,编码糖脂代谢过程中相关蛋白的基因,细胞周期相关基因,转录及翻译调节相关基因,应激反应相关基因,线粒体相关基因,细胞壁功能相关基因;显然与以往研究中报导的耐药基因的表达差异较大,通过实验发现氟康唑诱导SC5314由敏感株到耐药菌株的过程中,基因表达差异较大的是转录及翻译、代谢、蛋白酶体的基因;细胞耐药性的形成是多个基因共同作用,并且可能作用方式不同而导致的,可能不是几个单一的基因导致了耐药性的形成,有待于以后进一步的研究。耐药前后及香莲外洗液处理前后变化的是编码应激反应家族蛋白的基因:HSP30,HSP60,TIF51.3;RPT3,RPT5,HCT5.3eoc,UBP6.3属于编码蛋白酶体基因一族,及CDC1,CGR1,CCN1属于编码细胞周期相关蛋白基因,PPR1,SWI1,MEF2,TUF1,TIF3基因编码涉及转录及翻译调节的相关蛋白;总体上说明香莲外洗液作用的基因主要还是和细胞应激相关基因,公认的耐药基因受到影响较小。对不同模式的香莲外洗液处理的耐药菌株研究发现,受影响的基因主要是编码热休克蛋白基因SSB1、MGE1,丝氨酸/苏氨酸蛋白激酶基因,编码KNS1、RPS13.3、RPS22、PUP2、YST1.exon2、RPL10E及RPL28.3f于蛋白酶体家族基因,编码负责调控转录及翻译基因PRT1、IPF776、GCD11、CAP1、TIF35、CAM1.exon2、EFT2、EFT3。对于香莲外洗液作用于氟康唑诱导耐药菌株和临床耐药菌株后基因表达的研究表明,香莲外洗液作用后,白念珠菌从由应激回复稳定状态,如当白念珠菌处于耐药状态时,热休克蛋白表达升高,香莲外洗液作用后热休克蛋白表达降低;香莲外洗液作用于菌株后能恢复细胞正常功能,丝氨酸/苏氨酸蛋白激酶活性恢复正常。同时改善细胞载荷,当细胞处于耐药状态时,细胞内部蛋白酶体家族功能增强,降解相关蛋白质,促进细胞对外环境的适应性,香莲外洗液作用后,细胞内环境稳定,应激反应相关蛋白质减少,蛋白酶体相关基因表达水平降低。当然最为关键的是香莲外洗液能改善耐药相关基因的表达,恢复细胞的敏感性。本研究主要发现菌株的耐药是一个多基因涉及的复杂的过程,并不是几个单一基因作用的结果,本研究中,公认的耐药基因未发现表达差异,涉及细胞耐药性形成的基因以及香莲外洗液作用的靶点在近年来的文献中均未发现类似报导;也许中药有其自身独特的作用靶点;总之,在本实验中香莲外洗液体现出其独特的优势,不论是抑制念珠菌的生长方面,还是基因靶点方面,均和以往文献结果不同,值得以后详细的研究。更多还原

【Abstract】 ObjectiveAlong with the clinical use of azole drug in treating candidal vagin itis,the azole drug bears the developing resistance,the mechanism of fl uconazole to Candida albicans lies in the medicine enteing in the cell an d correlates with gene or some of key enzyme to play the roles of killing or suppressing the strain,lts drug resistance production also is connect ed the relative medicine gene expression or the variation result;It is d ifficult for patients to recover,already brought to the widespread atten tion;How to reduce the patient to azole drug resistance,or develops the treatment t to candidal vaginitis the new method,or the new medicine,r educes the patient pain,already was the major issue which the general me dicine worker faced.The research discovered that,the effect of clinical Chinese medicine treatment regarding candidal vaginitis is good,may redu ce the patient pain obviously;According to beforehand research,the effe ct of Xianglian solution treating the candidal vaginitis is remarkable,a nd has the obvious inhibitory action to the Candida albicans.The research about the resistance to azole drug of Candida albicans a lready arrived the molecular level under the modern molecular biology tec hnology,and in the unceasing,the scientists overseas already located to special genes now,and carry on the multi-positions research to special gene;Domestic research at present basically emphatically in whole functi on gene research,and elaborating the whole change of genes to the mechan ism of resistance.This goal of the research mainly appeals tothe modern molecular biolo gy technology,reveals the molecular mechanism of Xianglian solution in t he gene level to Candida albicans,including Candida albicans sensitive a nd resistance strain,analysis different strain of the function gene expr ession under the outside cleaning solution,carries on multi-target super elevation flux screening using the gene analysis correlation technology t o the Chinese native medicine wash-out side,constructs Xianglian solutio n gene function mechanism;The effect of Chinese medicine treating to be good,but its concrete action mechanism is obscure,has the similar actio n mechanism equally with fluconazole? has own specificity revelation targ et spot?Regarding repeatedly candidal vaginitis and the strain Candida albica ns the external use of Xianglian solution has good effect,what is it in the molecular action level mechanism also? It is worth our well research, provides the evidence for the Chinese medicine preparation standardizati on;It is significant for the research of Chinese medicine and moves towa rds the world.Method1 Fluconazole induces the SC5314 standard strain to resistance strain for the short-term,and observe the gene difference expression.Compare the resistance strain of short-term and the original generation of strain, understands the drug resistance in the gene stratification plane formati on.2 Xianglian solution was used to the SC5314 short-term resistance str ain,and observe the gene difference expression.Compare the two results, one is the difference of short-term SC5314 resistance strain versus the strain was used by Xianglian solution,the other result is the difference SC5314 standard strain to resistance strain for the short-term,by the c ontrast,we can know the detail spots on the function of the Xianglian so lution and can analysis the genes deeply.3 Compare the result about the laboratory induction strain used by Xia nglian solution and clinical resistance strain used by Xianglian solution. And analysis the result,to find the common difference genes,and discov er the function special target and carries on the multianalysis.4 Compare the uniform of NCCLS M27-A3,the S3 MIC observation method and the glucose consumption trial survey MIC method research.Uses the glu cose consumption trial to survey the MIC of Candida albicans processed by Xianglian solution.Result1 We may know the result of MIC of fluconazole to three kind of strai n,Candida albicans strain SC5314 MIC is 0.4883mg/ml processed by Xiangli an solution.The fluconazole function in SC5314 MIC is 0.25μg/ml;We may in fer in vitro experiment of suppressing Candida albicans SC5314,Xianglian solution 0.4883mg/ml and fluconazole 0.25μg/ml are equal.2 Along with Xianglian solution density rise,the experimental two ki nd of strain various glucose density is higher,explained the glucose con sumption are less,after taking the logarithm of the medicine density and carring on the analysis,we discovered the logarithm of the medicine den sity and each glucose density presents the linear relations;Compare the two kind of strain linear relations,and found the uniformity to two kind of strain functions.Along with the fluconazole solution density rise,the experimental two kind of strain various glucose density is higher,showe d the glucose consumption are less,takes the logarithmof the medicine de nsity,discovered the logarithm the medicine density and each glucose den sity presents the linear relations;Carries on the analysis comparison to under the fluconazole function two kind of strain linear relations,disc overed the relations of the two strains has the nonuniformity.3 Compare the fluconazole induction reaiatance SC5314 strain and sens itive strain compares,its difference gene expression mainly concentrates in:The code proteinase body and the protein hydroltyic enzyme gene,in the code sugar fat metabolism process is connected the protein gene,the cell cycle correlation gene,the duplication and the translation adjustme nt correlation gene,the stress response correlation gene,the line plast ochondria correlation gene,the cell wall function related gene.4 Candida albicans SC5314 induction resiatance strain was processed b y Xianglian solution,its expression change gene mainly is:Code stress re sponse family protein gene,biomembrane relevant gene,a code proteinase body gene race,code cell cycle related protein gene,duplication and tra nslation adjustment related protein gene.5 The clinical reaiatance strain Candida albicans was processed by Xi anglian solution,its expression change gene mainly is:Codes the hot sho ck protein gene,the serine/threonine protein activating enzyme gene,the proteinase body family gene,the regulation copies and translates the ge ne.ConclusionXianglian solution,takes one kind of hospital self-restraint externa 1 use medicament,although its development utilization has about 10 years, but also only is limits to the hospital use,the Xianglian solution not yet starts regarding Candida albicans MIC research,therefore is also far the standard criteria MIC to Candida albicans.Therefore,this experimen t has also carried on the preliminary exploration to Candida albicans of the Xianglian solution MIC determination,provokes the thought for later research.In this experiment,Xianglian solution in experimental strain S C5314 its MIC is 0.4883mg/ml.The fluconazole function in SC5314 MIC is 0. 25μg/ml,We can basically infer in vitro experiment Candida albicans SC53 14,Xianglian solution 0.4883mg/ml and fluconazole 0.25μg/ml have the equ al potency.Xianglian solution and fluconazole to the Candida albicans glucose co nsumption influence and the analysis research discovered Xianglian soluti on has the uniformity to two kind of strain functions.Along with the flu conazole solution density rise,the experimental two kind of strain vario us glucose density is higher,the explanation glucose consumption are les s;Carries on the analysis comparison to under the fluconazole function t wo kind of strain linear relations,discovered that the two strains have the nonuniformity.This possible to explain two medicines the function mec hanisms are dissimilar,fluconazole main function in cytochrome P450 ster ol synzyme way,but Xianglian solution function is unknown actually in an y way;Perhaps this is Xianglian solution and the fluconazole function un similarity,will wait for further studies in later.Observation of the fluconazole resistance strain SC5314 and sensitive strain carring on the gene chip research discovery,its difference gene expression mainly concentrates in:The code proteinase body and the prote in hydroltyic enzyme gene,in the code sugar fat metabolism process is co nnected the protein gene,the cell cycle correlation gene,the duplicatio n and the translation adjustment correlation gene,the stress response co rrelation gene,the line plastochondria correlation gene,the cell wall f unction related gene;Obviously with formerly studied reports about the r eaiatance genes the expression was difference,through this experiment di scovered fluconazole induced SC5314 to bear the medicine strain from sens itive in the process,the gene expression difference was the duplication and the translation,metabolism,the proteinase body gene;The cell drug resistance formation is many genes affects together,and possible to affe ct,possibly is not several sole genes which the way different causes has caused the drug resistance formation,will wait for in the later further research.Campare the result of the sensitive to resistance strain and the stra in was processed by Xianglian solution,changes gene is codes the stress r esponse family protein the gene:HSP30,HSP60,TIF51.3;RPT3,RPT5,HCT5. 3eoc,UBP6.3 belongs to a code proteinase body gene race,and CDC1,CGR1, CCN1 belongs to the code cell cycle related protein gene,the PPR1,SWI1, MEF2,TUF1,TIF3 gene code involves the duplication and the translation adjustment related protein;As a whole explained Xianglian solution funct ion gene mainly with the cell stress correlation gene,the recognition be ars the medicine gene to come under the influence to be small.The observation of the different resistance strains processed by Xian glian solution,we discovery the gene which affects mainly is codes hot s hock protein gene SSB1,MGE1,the serine/threonine protein activating enz yme gene,codes KNS1,RPS13.3,RPS22,PUP2,YST1.exon2,RPL10E and RPL28. 3f in the proteinase body family gene,the code is responsible to regulat e copies and translates gene PRT1,IPF776,GCD11,CAP1,TIF35,CAM1.exon2, EFT2,EFT3.The research of Xianglian solution affecting fluconazole inducing str ain and clinical reaistance strain indicated that after Xianglian solutio n processing,Candida albicans come from stress to steady state,when the Candida albicans is in bears the medicine condition,the heat is in shoc k the protein expression ascension,Xianglian solution function secondary fever shock protein expression reduces:Xianglian solution function can restore the cell normal function after the strain,the serine/threonine p rotein activating enzyme revivification is normal.Simultaneously improve s the cell load,when the cell is in bears the medicine condition,the ce ll internal proteinase body family function strengthens,degrades the cor relation protein,the promotion cell foreign environment compatibility,a fter Xianglian solution function,in the cell the environment is stable, the stress responded the related protein reduces,the proteinase body cor relation gene expression level reduces.Certainly most essential is Xiang lian solution can improve bears the medicine correlation gene the express ion,restores the cell the sensitivity.This research main discovery strain bears the medicine is the complex process,is not several sole gene function result,in this research,the recognition bears the medicine gene not to discover the expression diffe rence,involved the gene as well as Candida albicans which the cell drug resistance formed the cleaning solution function target in the recent yea rs literature has not discovered the similar report;Perhaps the Chinese medicine has its own unique function target spot;In brief,Xianglian sol ution appears its unique superiority in this experiment,no matter is sup presses the Candida albicans the growth aspect,a gene target aspect,and the former literature result is different,is worth later detailed resea rch.更多还原