Study Of The Inhibitory Effects On Candida Albicans Of The Vagina Cells Transferred With LL-37and HD5Recombinant Plasmids

Vulvovaginal candidiasis (VVC) is a widespread and common disease affecting a large proportion of women of all ages. Approximately75%of women experience at least one episode of VVC during their lives, most commonly when they are childbearing age. The incidence of VVC has dramatically increased over the past few decades due to the abuses of broad-spectrum antibiotics, the using of contraceptive agents and vaginal irrigation. Unfortunately, there has not been a correspondingly rapid development of therapeutics to combat with drug resistant. Since it is already obvious that the currently known antibiotics and possible derivatives will sooner or later lose their efficiency, the search for novel targets and chemicals is of great value. Antimicrobial peptides (AMPs) have a broad-spectrum anti-microbial properties and non-toxic, non-resistance, which have become hot factors for anti-infection studying.Objective:To evaluate the inhibitory effects on Candida albicans of the vagina cells transferred with antimicrobial peptide LL-37and HD5recombinant plasmids and observe secretion of chemokine IL-8.Method:(1) Total RNA was extracted from human vaginal epithelial cells. The cDNA encoding the LL37and HD5were amplified through RT-PCR. which were inserted into pcDNA3.1(+)-EGFP, an expression vector.(2) The vaginal epithelial cells from female vagina were cultured primarily by fractional step digestion method and serum-free keratinocyte medium. The Candida albicans stain ATCC90028was cultured in vitro and prepared as bacteria suspensions.(3) The pcDNA3.1(+)/HD5-EGFP and pcDNA3.1(+)/LL37-EGFP recombinant plasmids were separately or jointly transferred into the fourth passage of vaginal epithelial cells. Two test groups were defined:one test group was no Candida albicans group including four subgroups which were untransferred subgroup, HD5subgroup transferred with pcDNA3.1(+)/HD5-EGFP, LL37subgroup transferred with pcDNA3.1(+)/LL37-EGFP, combining transferring subgroup transferred with pcDNA3.1(+)/HD5-EGFP and pcDNA3.1(+)/LL37-EGFP. The other test group was with Candida albicans group which the Candida albicans were coincubated with the four subgroups described above. The ration of the vaginal epithelial cells and Candida albicans was40:1. The supernatant of every group was collected and ELISA was applied to detect the level of LL37, HD5and IL-8at6,12,24and48h. At each time point, the growth density of Candida albicans was observed and the growth inhibition rate was calculated by glucose consumption testing.Result:(1) Our study constructed pcDNA3.1(+)/HD5-EGFP and pcDNA3.1(+)/LL37-EGFP recombinant plasmids.(2) The cells cultured in vitro were typical vaginal epithelial cells without contaminated by fibroblasts during the whole growth phase. Immunohistochemical staining using keratin and counting by microscope demonstrated that the purity of vaginal epithelial cells was99%. The trypan blue staining showed the cell viability reaching95%to99%. The cells could be subcultured for7～9passages and live up50～60days in vitro. The Candida albicans colonies were well developed in vitro and could form spore and pseudohypha.(3) The LL-37and HD5recombinant plasmids were successfully transfected into human vaginal epithelial cells, which the transiently transfected rate could reach to100%. The secretion level of LL-37. HD5and IL-8reached the max level after transferred for24hours, then showed decreasing trend. The secretion level of LL37, HD5and IL-8were significant higher in combining transferring subgroup with Candida albicans than other groups, and the expression of LL37, HD5and IL-8were separately (100.16±0.81) ng/ml,(58.50±2.08) ng/ml and (101.03±1.59) pg/ml (P<0.01). Following the6h coculture with Candida, non-transfected subgroup showed the increase in Candida growth density and decrease in the number of epithelial cells with incubation time extending, while LL37subgroup. HD5subgroup and in particular co-transfected subgroup showed no significant growth of Candida and the epithelial cells survival rate was higher. In different time point, the absorbance of each subgroup without Candida albicans declined slowly and there were no statistically significant difference (P>0.05), as while as in LL37subgroup and HD5subgroup with Candida albicans in same time point(P>0.05). In group with Candida albicans, the absorbance of combining transferring subgroup were3.210±0.010,3.150+0.030,3.099±0.030and2.970±0.040at6.12,24and48h, respectively, which were significantly higher than those in the other cells (P<0.01) and the declined trend was the slowest.Conclusion:(1) Recombinant plasmids of pcDNA3.1(+)/LL-37-EGFP and pcDNA3.1(+)/HD5-EGFP with fluorescent protein were successfully constructed.(2) The fractional step digestion method and serum-free keratinocyte medium were good choice for the primary culture of vaginal epithelial cells. The primary cultured cells viability, survival and proliferative abilities were well, and the purity was high. After transfer of culture, the vaginal epithelial cells remained good reproductive potential, which provided an excellent experimental model for studying the vaginal mucosal disease in vitro.(3) The LL-37and HD5recombinant plasmids were transfected into vaginal cells to highly express LL-37and HD5protein, which were found to be biologically active and promoted expression by each other. The antifungal ability of vaginal epithelial cell became stronger after being transferred with LL-37and HD5recombinant plasmids. LL-37and HD5could also possess immunomodulatory activity and induce chemokine IL-8production. Our research offered reliable testing information for genie treatment of VVC in future.