A Comparative Study Of The Invasiveness Of RVVC Pathogenic Strains To Murine Vaginal Epithelial Cells In Vitro

Objectives:1.To obtain high purity, good activity and stability batches three to four generations of mouse vaginal epithelial cells by improving the method of culturing mouse vaginal epithelial cells in vitro.2. To identify the strains of pathogenic yeast in Vulvovaginal Candiddiasis (VVC) and Recurrent Vulvovaginal Candiddiasis (RVVC), and to investigate in vitro fluconazole resistance.3.To analyze invasiveness of the pathogenic yeasts bearing different fluconazole susceptibility to murine vaginal epithelial cells in vitro amony in the mechanism of RVVC, and lay a foundation to make a further research in the pathogenic mechanism of RVVC in vitro. Methods:1. Vaginae from female mice were excised under aseptic conditions and separated by dispase â…¡-neutralprotease (Dispase II) and trypsin digestion method, cultured with Eplife medium and subcultured the cells when its convergence degree reached 80-90%. Then dynamically observed the cell growth conditions under optical microscope, cell surface and morphology under scanning electron microscope (sem), cell with Pan-CK, CK.5/6 antibody immunohistochemical to identify cell sources.2. A total of 406 Candida isolates recovered from VVC and RVVC patients in the clinic were identified with CHROMagar chromogenic medium, the chromogenic medium of Autobio and the VITEK 2. A modifying microdilution fluconazole susceptibility test was preformed.3. One from each VVC type and fluconazole susceptibility category (susceptible (S); susceptible dose dependent (SDD); resistant (R)), Candida strain was selected, and co-cultured with vaginal epithelial cells for 6h,12h and 24h. The appearance and inner micro-construction of co-cultured cells was observed through scanning electron microscope and transmission electron microscope respectively at different times.Results:1. Most of the original generation of vaginal epithelial cells post 24h enzyme digestion, settled to the bottom of bottle as a small spherical water-drop with poor refraction.Then cell tentacles extended gradually and the cell body became flat over time with higher reflection.During the early stages of culture progcess,cell was water-drop with stubby tentacles and cell body bulged out under scanning electron microscope. Cells tentacles then became slender and cell body flattened.With increase in the density, cell edge become thickened forming a clear cell line, arranged like paving stone. Pan-CK, CK5/6 keratin monoclonal antibody immunohistochemical staining was positive.2. In total of 406 strains of pathogenic candida from RWC and WC patients,Candida albicans of VVC accounted for 86.31% and 13.69% was non-albicans; Candida albicans of RVVC accounted for 90.91% and 9.09% accounted for non-albicans; The constituent ratio of Candida albicans and non-albicans in two groups has no significant difference, P>0.05. Overall, fluconazole sensitive Candida albicans accounted for 97.5%(349/358) in 24 hours. In VVC group, 98.6%Candida albicans was sensceptible to fluconazole and none was resistant.In RWC group,96.0% Candida albicans was sensceptible and 8% was resistant.The overall fluconazole susceptiblity rate for non-albicans in 24h was 87.5% (42/48), Non-albicans in VVC group showed 84.9% susceptiblity and 6.1% resistence rate. As for non-albicans in RVVC group,93.3% was susceptible and 6.7% was resistant. No difference was found in fluconazole susceptibility of Candida albicans and non-albicans in both VVC and RVVC group,,P>0.05.3.Pathogenic candida from VVC and RWC cultured with vaginal epithelial cells for 6h,12h and 24h, did not show significant difference between the fluconazole sensitive and dose dependent strains amony RVVC and VVC group, fluconazole resistant RVVC and VVC strains did not differ in 6 h, but the destruction capacity in12h was significantly greater in RWC strainsin.Hyphal phase cell adhesion to vaginal cells and cell morphology was incomplete under scanning electron microscope.After the cell was invaded by yeasts, nuclear membrane continuity was interrupted, intracellular organelles and "cotton wool "necrosis was observed under transmission electron microscope.Conclusions:1.The original generation of murine vaginal epithelial cells to culture in vitro in a short time was accomplished and obtained high purity, good activity of vaginal epithelial cells, which laid a foundation to study the role of vaginal local immunity in Recurrent Vulvovaginal Candidiasis.2. Candida albicans is the dominant isolate in VVC and RWC, accounting for about 80-90%, the ratio of non-albicans in WC patients has a tendency to rise year by year. Compared with the previous reports, fluconazole resistance phenomenon is not so serious, a majority of RWC, WC pathogenic strains are sensitive to fluconazole.3. Relative to the VVC, RWC fluconazole resistant strains showed more aggressive invasiveness on vaginal epithelium in vitro, the different invasiveness associated with pathogenic strains resistance may be the important reason for RVVC recurrence and treatment failure.