【作者】 佘晓东；

【导师】 刘维达； 陈沄； 李岷； 吕桂霞；

【作者基本信息】 中国协和医科大学 ， 皮肤性病学， 2008， 博士

【摘要】 外阴阴道念珠菌病(vulvovaginal candidiasis,VVC)是一种由念珠菌(属)引起的阴道粘膜和或外阴的真菌感染。对于该病的发病机制,国内外学者从不同角度进行研究,得到了各异的研究结果。前些年国外学者经过研究几成共识的是:在阴道念珠菌病,是局部而非系统的体液免疫和细胞免疫对真菌感染呈保护作用,而且天然免疫可能参与了局部的免疫作用。此外,念珠菌在致病条件下的毒力增强也是该病的一个重要发病机制,而且这种菌株毒力特性可能与其特殊的基因型有关。可见,涉及本病发生发展的因子和因素很多,舍弃某一个研究方向都有可能遗漏重要的致病机制。因此,在本次研究中我们想到应用不同的研究方法针对不同的可能致病因素进行分析。借助微点阵芯片、荧光定量PCR等技术的高通量、高灵敏度的优势,从各相关因子在疾病不同阶段和不同状态时表达的高低和动态变化,来整体地系统地较全面地分析探讨本病的发病机制,试图揭示宿主和病原菌相互作用的内在关系,以期得到更接近于真实的结果,为今后临床更有效地防治本病打下良好基础。第一,利用自制Oligo基因芯片研究小鼠念珠菌性阴道炎的发病机制。首先,成功构建雌激素化小鼠阴道念珠菌病模型,并设立单独雌激素化小鼠阴性对照组以及正常小鼠空白对照组。其次,根据需要自行设计制备小规模Oligo基因芯片,将不同组别的模型标本与芯片进行杂交,并将表达信号差异超过2倍的因子进行筛选与分析。研究结果显示,在宿主免疫方面,炎性趋化因子普遍表达增高,适应性免疫调节因子IL-1、IL-4、IL-6、IL-10、IL-12、TNF-α、NF-κB以及TGF-β均有不同程度增强,其中以IL-1和TGF-β增强最为明显。全部标本天然免疫成分TLR4的表达增高,TLR2仅表达增高于1/3的标本。在致病菌株毒力方面,EFG1、SAPs2、4、5、6、10、LIP2、LIP4和HWP1表达显著增强。实验结果说明,在小鼠阴道局部免疫过程中,细胞免疫较体液免疫在发病机制中似乎占有更重要的地位,但两者表达增高均不具有普遍现象,适应性免疫或许不能作为阴道念珠菌病的主要免疫机制。天然免疫成份TLR4可能参与了阴道念珠菌病患者的局部免疫过程。在致病菌株毒力方面,由EFG1通路介导的菌株表型转换、菌丝生成以及毒力增强是念珠菌性阴道炎发病的另外一个重要因素。第二,利用Oligo基因芯片对念珠菌性阴道炎患者阴道分泌物标本进行研究。首先,探索念珠菌性阴道炎患者阴道分泌物标本的RNA提取方法。通过对匀浆、玻璃珠和液氮研磨三种破壁方法,纯化柱、酚两种提取方法组合产生的5种不同RNA提取方法进行比较,发现以液氮研磨后Trizol提取法效果最好,可实现两种不同成分的共同提取,为直接应用临床标本进行芯片研究奠定了基础。然后,使用自制疾病相关因子Oligo芯片对疾病相关因子进行研究。结果显示,在阴道念珠菌病发病过程中,炎性细胞趋化因子普遍表达增高。适应性免疫因子部分表达增高,其中在VVC患者中Th2型细胞因子表达占优,而RVVC患者中Th1和Th2细胞因子无明显差别;但上述表达增高因子不具有普遍性,且部分因子表达不稳定。天然免疫因子TLR4表达普遍增高,而TLR2表达增高不显著。在致病菌株毒力方面,EFG1、CPH1、SAP2、SAP5、SAP6、SAP8、LIP4和HWP1等因子表达增强。综合人体临床标本以及动物模型实验结果显示,LIP4、SAP2、SAP5、HWP1、RANTES、MCP-1、MIP-1α、MIP-2、IL-1α、NF-κB、TLR4等因子在两种研究对象中均表达显著增高。实验结果说明,适应性免疫参与了患者阴道局部的部分免疫,而且体液免疫在VVC患者中占有一定优势,在RVVC患者中两种免疫成分具有相同的作用,但这些免疫成分可能作用有限。由TLR4介导的天然免疫反应可能参与了阴道组织对念珠菌感染的免疫反应。另外,由EFG1和CPH1通路共同介导的表型转换、菌丝生成以及毒力增强参与了致病菌株的致病过程,是阴道念珠菌病发病的另外一个重要因素。在两种研究对象中均表达增高的致病因子可能与疾病发生高度相关,而值得今后深入研究。同时,这也佐证了所采用的小鼠模型完全可以替代人体实验进行本病的一些深入探讨研究,如体内药效学评价、疫苗免疫、致病相关基因(如TLR4)敲除等。第三,使用荧光定量PCR方法检测念珠菌性阴道炎患者阴道分泌物的天然免疫成分。利用荧光定量PCR技术的高灵敏性、高特异性的特点,将最近研究较多的天然免疫成分包括防御素(β-defensin-2)、甘露糖凝集素(MBL)、Toll样受体家族等天然免疫成分进行定量检测分析,通过对比不同发病状态时各天然免疫成份的表达变化研究其在发病各个阶段所起的作用。结果显示,β-defensin-2可以表达于除1例VVC患者之外的全部患者标本中,而在健康带菌者标本中未表达,而且表达量在VVC和RVVC患者分泌物标本中均显著升高。MBL几乎在全部分泌物标本中可以被检测到,但在三组标本间未发现该基因表达水平的差异。TLR1、TLR2、TLR s4-6、TLR8表达于所有标本中,TLR9、TLR10部分表达于三组标本中,而TLR3和TLR7则仅见于部分感染标本中。定量结果显示,健康带菌者TLR3和TLR4表达水平较两组患者均明显降低,TLR2的表达水平较RVVC患者降低,其余TLRs均未见明显差异。实验结果说明,β-defensin-2介导的免疫反应可能由白念珠菌的菌丝相诱导参与了机体的抗白念珠菌感染作用。而MBL在机体由带菌状态转变为致病状态或者复发状态的过程中未起明显的免疫作用。在TLRs家族中,TLR3和TLR4可能在阴道念珠菌病发病过程中有较为重要的免疫机制,而TLR2的作用尚不清晰。第四,使用PCR技术对分离自阴道念珠菌病以及皮肤念珠菌病的白念珠菌进行基因型的比较。通过PCR技术扩增白念珠菌染色体25S rDNA片段和RPS序列片段,对分离自阴道念珠菌病患者以及皮肤念珠菌病患者的151株白念珠菌进行基因分型。结果显示,阴道分离菌株和皮肤分离菌株中A和Ⅰ基因型为分离菌株的主要基因类型,而C和Ⅴ型为少数基因型。Ⅳ和Ⅴ型菌株仅发现于皮肤念珠菌病患者。比较两组患者分离菌株各基因型比例显示,除C型外,两组间各基因型分布均存在显著性差异。而且具有更强毒力的A型白念珠菌在阴道念珠菌病分离菌株中明显增多。实验结果表明,阴道粘膜和皮肤不同的定植环境可能会影响感染菌株的基因型,而且阴道感染菌株具有更强的毒力,这也可能是阴道念珠菌病发病的另一个因素。总结:我们对念珠菌性阴道炎的发病机制从宿主免疫和菌株毒力两个研究方面,以及从mRNA表达差异和DNA遗传差异两个层面进行了研究。在宿主免疫方面,未见国外研究报道的局部细胞免疫缺陷现象,但各种适应性免疫成分表达总体偏低,适应性免疫可能功能有限而不能作为阴道念珠菌病的主要免疫机制。相反,天然免疫成分可能作为机体应对局部念珠菌感染的重要免疫介导因子,其中β-defensin-2、TLR3和TLR4在阴道念珠菌病发病的免疫机制中有较为重要的作用。在菌株毒力表达方面,由EFG1和CPH1通路介导的表型转换,以及SAPs、LIPs和HWP1表达增强导致菌株毒力增强是念珠菌性阴道炎发病的另外一个重要因素,而且这种菌株毒力增强可能与其特定的基因型遗传特征有关。总之,适应性免疫的功能有限以及菌株的毒力增强这两方面的因素均参与了念珠菌性阴道炎的发病机制。更多还原

【Abstract】 Vulvovaginal candidiasis (VVC) is a common gynecological disease in childbearing women caused by Candida spp. The studies concering the pathogenesis of VVC are numerous, but the results are different or even contradictory. So, its pathogenesis is still unclear. Now, it is commonly accepted the theory that there are local but not systemic humoral immunity and cell immunity in VVC and innate immunity may be a key protective mechanism. In addition, the enhancements of the virulence of the pathogenic strains were also possible pathogen factors and the different virulence between strains may be related to their genotypes. So, the pathogen factors related to VVC are numerous and the study in one aspect maybe omits some other important pathogen factors. In this study, we utilized different methods researching different possible pathogens. With the superiority of the high capacity and high sensitivity from gene chip and real time PCR, we investigate all the possible pathogen factors in one unique condition and analyze the pathogenesis of VVC systemically. The results of our research may make a preliminary foundation for the prevention and treatment of VVC.Part I. Using oligo gene chips to investigate the pathogenesis of vulvovaginal candidiasis in mouse model.Firstly, mice models of vulvovaginal candidiasis and two control groups for research were established. Secondly, Oligo gene chips including the possible factors from host and pathogenic strains were conducted. Finally, RNA mixtures from different mouse model group and Candida albicans (C. albicans) strains were hybridized with the gene chip and the factors which signals increased or decreased two folds were selected and analyzed. The results showed that local cell immunity was more important than humoral immunity, but both were not increasing significantly during the infection. Whereas, the innate immunity component TLR4 incresed obviously in infective samples. On the other hand, the expression of virulent factors EFG1, SAP2, SAP4, SAP5, SAP6, SAP10, LIP2, LIP4 and HWP1 increased in pathogenic strains. The research indicated that the adaptive immune was not the important immunity in VVC of mouse model, but the TLR4 which mediated the innate immune maybe play a key role in the immunity of the host. In addition, the enhancement of virulence of pathogenic strain mediated by EFG1 pathway play an important role in the pathogenesis of VVC.Part II. Using oligo gene chips to investigate the pathogenesis of vulvovaginal candidiasis in patient samples.First, a simple and rapid method of RNA extraction for vaginal secretion from patient with VVC was investigated. Comparing extraction quality of 5 methods for RNA extraction of the mixture of standard strain of C. albicans and HaCaT cell line showed the method of TRIzol after grinding with liquid nitrogen was the best method which could extract RNA from Candida spores and human cells. Second, the RNA of vaginal secretion from patients with VVC in different infect conditions were hybridized with the Oligo gene chips. The results showed adaptive immunity components were found in the vaginal secretion of patients. Furthermore, the humoral immunity were superior in the VVC patient, the cell immunity and humoral immunity were equal in RVVC patients. But the enhancements of adaptive immunity factors in were not universal. The expression of TLR4 incresed significantly in patient samples, but the expression of TLR2 was instable. Similar to the results of mice models, the expression of virulent factors EFG1, CPH1, SAP2, SAP5, SAP6, SAP8, LIP4 and HWP1 increased in pathogenic strains. The results of this study indicated that effects of adaptive immune were limited because of their low expression. The innate immune mediated by TLR4 maybe important in the course of VVC. In addition, the enhancements of virulence of pathogenic strains mediated by EFG1 and CPH1 co-pathway were important in the pathogenesis of VVC. Finally, the pathogen factors LIP4, SAP2, SAP5, HWP1, RANTES, MCP-1, MlP-1α, MIP-2, IL-1α, NF-κB and TLR4 both involve in the onset of VVC in mice and human, which seem highly related to the disease and need a further investigation.Part III. Using real time PCR to investigate the components of innate immunity in patient samples.In this study, the expression of innate immunity components includingβ-defensin-2, MBL and TLRs in vaginal secretion from different patient groups were investigated by real time PCR, and the effect of each immunity component was evaluated after comparing its expression variation in different infective conditions. The results showed that except one VVC and all carrier samples, P-defensin-2 could be found in other patient samples and its expression in patient samples was obvious higher than carriers. MBL could be found in almost all the samples, but no significant differences in expression levels among three groups were found. The distribution of TLRs showed that TLR1, TLR2, TLRs4-6, TLR8 were found in all the samples, TLR9 and TLR10 were found in part samples, whereas, TLR3 and TLR7 were only found in part samples of VVC and RVVC patients. Expression of TLR3, TLR4 and TLR2 in carrier samples decreased significantly comparing with VVC and RVVC patients. The research demonstrates that the innate immunity mediated by P-defensin-2 participates in the course of anti-infection, whereas MBL might not play an important role in the transition of carriers to VVC or RVVC. In addition, TLRs2-4 but not the other TLRs participate in the local immunity of vaginal in the course from the colonisation to infective condition.Part IV. Investigating the possible association between genotypes of the Candida albicans isolates and vulvovaginal candidiasis.In this study, 151 strains of C. albicans isolated from 74 infant patients with cutaneous candidiasis and 61 female patients with vaginal candidiasis were genotyped by using 25S rDNA and RPS-based PCR. Ten genotypes were detected and obvious differences in frequencies of genotypes between two groups were found, especially in genotype A and B. Furthermore, the number of genotype A strain with more virulence was significant higher in vulvovaginal candidiasis group. The results indicate that the obvious different environments of cutaneous and vaginal mucous membrane for C. albicans colonizing maybe influence the genotypes of invasive strains, and the C. albicans strains from vagina with more virulence which may be another pathogen for VVC.Summary: We investigated the pathogenesis of VVC from two aspects including host and pathogenic strains and in two levels of mRNA and DNA. The results of series research suggest that the deficiency of adaptive immunity and the increased virulence of pathogenic strains played an important role in the pathogenesis of VVC. The innate immunity components P-defensin-2, TLR3 and TLR4 were important in the immunopathogenesis of VVC and the evaluated virulence of pathogenic strains may be related to their specific genotypes.更多还原