The Preliminary Investigation On The Pathogenesis Of Vulvovaginal Candidiasis By Oligo Gene Chip & PCR

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.