| Candida species are commensal fungal organismsas and opportunistic pathogens, while Candida albicans is the main cause of human oral and vaginal mucosa infection pathogens.Although Oral and vaginal mucosa epithelial cells are the first line to defend against Candida infection, there are similarities and differences in the immune response of epithelial cells infected by Candida albicans.The epithelial cells could activate the inflammatory signaling pathways (e.g., ERK/JUK/p38 and other signaling pathways) and secrete the inflammatory cytokines to defend Candida albicans. The cell wall components ALS3 and SSA1 of Candida albicans play are important for the induction of epithelial cells in the immune process. Previous studies have demonstrated that the knockdown of ALS3 can reduce the expression of inflammatory cytokines and inflammatory signal pathway proteins in oral epithelial cells, but there are few experimental studies on the inflammatory cytokines and the inflammatory pathways in vaginal epithelial cells infected by the ALS3 knock-out mutant, or in oral and vaginal epithelial cells infected by the SSA1 knock-out mutant.In this study, we investigated the differences of inflammatory cytokine production and signaling pathway activation in oral epithelial cells and vaginal epithelial cells when they were infected by Candida albicans. Meanwhile,we studied the immune differences in the cells of diverse sites infected by the ALS3 mutant and SSA1 mutant.Chapter I The differences of immunity between oral and vaginal epithelial cells infected by C.albicansObjective:To explore the similarities and differences of the immunity between oral and vaginal epithelial cells when they are infected by C.albicans in vitro.Methods:we cultured the oral and vaginal epithelial cells and establish the model of epithelial cells infected by C.albicans in vitro. And then we analyzed the differences by detecting the cytotoxicity, the secretion of cytokines and the role of MKP1 and ERK1/2 protein in the oral and vaginal cells when C.albicans infected.Results:There are some similarities existed in oral and vaginal epithelial cells:under inverted microscope, their morphology looks similar. They look like paving stones or polygons. After Wright Giemsa staining, Cells showed a typical pink cytoplasm and nuclei blue (black) color performance. About the Growth curve results, there are differences in the growth and proliferation of leuk-1 cells and VK2/E6E7 cells. The proliferation rate of Leuk-1 cells was faster than that of VK2/E6E7 cells. During the growth process, the diameter of the cells also changed. The diameter of the cells in the two groups showed a similar parabola shape in the process of growth and proliferation. But the whole diameters of leuk-1 was still larger than that of VK2/E6E7 cells. In the infected site, the dead cells were distributed along the hyphae. The results showed that the damage degree of the same kind of cells was related to the dose of infection of C.albicans and the time of infection, that is, the damage was proportional to MOI (multiplicity of infection) and time. In the comparison of the two groups of cells, the epithelial cell damage by C.albicans is site-specific. When infected by the same MOI of C.albicans, the damage of vaginal cell VK2/E6E7 is badly, and the damage of oral cell leuk-1 cell line is relatively less. The detection of cytokines showed that some cytokines,such as GM-CSF, G-CSF, IL-1α, IL-1β,MIP-3α, IL-8, and RANTES could be induced by C.albicans in both oral epithelial cells and vaginal epithelial cells. But there are also differences:while oral epithelial cells uninfected would produce a variety of cytokines, and even can produce more secretion in low doses of Candida albicans infection; in the vaginal epithelial cells uninfected, most of the cytokines are rare, and even in Candida albicans infection, the stimulation of most cytokine is weaker. In the two types of cells, the best initial doses which could stimulate the secretions of cytokins were different. In the oral infection, IL-8, GM-CSF, MIP-3α and RANTES showed their peak at MOI=0.01, although the peak of G-CSF and IL-1β appeared at MOI = 0.1 and IL-1α at MOI=1. While in the vaginal cells, IL-8, GM-CSF showed their peak at MOI=0.01, G-CSF at MOI=.1,and IL-1α, IL-1, MIP-3α at MOI=1. In the detection of IL-4 and IL-12 cytokines, it did not show obvious change when the cells were infected by C.albicans. About the expressions of the ERK1/2 and p-MKP1 proteins, we detected the oral and vaginal epithelial cells after infected by C.albicans for 2 hours:the protein expressions of the infected group were higher than uninfected group,and the difference is statistically significant.Conclusion:when Candida albicans infected different epithelial cells cells, the cells damage and immune cytokines is not the same. There was site-specific.Chapter Ⅱ The role of ALS3 in oral and vaginal epithelial cells infected by C.albicansObjective:To explore the similarities and differences between the immunity stimulated by SC5314 (wild type) and the ALS3 mutant of C.albicans in oral and vaginal epithelial cells.Methods:we respectively cocultured the oral and vaginal epithelial cells with the wild type SC5314 and the ALS3 mutant in vitro. Then we explored and contrasted the differences of the immune related factors in different groups infected by different strains in the same cell type or betwwen the two cell types, such as cell toxicity, cytokines, the expressions of p-ERK1/2 and p-MKP1 pathway proteins.Results:Compared with the wildtype, ALS3 mutant did not affect the formation of hyphae, but its cell damage ability was significantly lower. The cell damage abilities of the wild type and the mutant were both dose-dependent infection. The bigger the MOI was, the greater the damage was. When infected by ALS3 mutant in the oral cells, the expressions of GM-CSF, G-CSF, IL-1α, IL-1, MIP-3α, IL-8 were lower than WT, while in the vaginal cells, there were GM-CSF, G-CSF, IL-1,IL-8. Compared the differences when the same strain infecting the two types of cells, we found the ALS mutant could eliminate the differences in two cells in the expression of G-CSF, RANTES, MIP-3α and IL-8. When oral epithelial cells infected by C.albicans for 2h, the expression of p-ERK1/2 protein in WT group and that in the ALS3 mutant group were the same, while the expression of p-MKP1 infected by WT were higher than that infection by ALS3 mutant. In vaginal cells VK2/E6E7, the expression of p-ERK1/2 protein and p-MKP1 protein infected by ALS3 mutant were much lower than that by WT.Conclusion:ALS3 can induce the expression of a variety of immune effector in oral and vaginal epithelial cells. In the oral cells, ALS3 do not activate the expression of p-MKP1 protein to induce immunity entirely through the ERK immune signaling pathway, while in the vaginal cells, ALS3 induces the expression of p-MKP1 protein exactly through activating ERK pathway..ALS3 may be one of the possible factors that produce the difference of immune in the vaginal epithelial cells and oral cells.Chapter Ⅲ The role of SSA1 in oral and vaginal epithelial cells infected by C.albicansObjective:To explore the similarities and differences between the SC5314 (WT) and the SSA1 mutant in the induction of immunity in oral and vaginal epithelial cells.Methods:we cocultured the wild type and the SSA1 mutant of C.albicans with the oral and vaginal epithelial cells respectively in vitro. Then we explored and contrasted the differences of the immune related factors in different groups infected by different strains in the same cell type or betwwen the two cell types, such as cell toxicity, cytokines, the expressions of p-ERK1/2 and p-MKP1 pathway proteins.Results:SSA1 mutant affect the formation of hyphae. Its hyphae were shorter, and the cell damage ability of the mutant was significantly lower.The cell damage infected by mutant was also dose dependent. The bigger the MOI value was, the greater the damage was.When the oral cells infected by SSA1 mutant, the expressions of GM-CSF,IL-1α, IL-1β, RANTES, MIP-3 a, IL-8 were lower than that by WT, while in the vaginal cells, there were G-CSF, IL-1 α, MIP-3 α, IL-8. Comparative analysis of the differences when the same strain infecting the two kinds of cells,the mutant could eliminate the differences in IL-1 β ,RANTES, MIP-3 α, IL-8. In oral epithelial cells infected by C.albicans for 2h, the expression of p-ERK1/2 protein in WT was higher than that of SSA1 mutant; in contrast, the expressions of p-MKP1 protein in WT group were the same with that in SSA1 mutant group. In vaginal cells VK2/E6E7, the expressions of p-ERK1/2 protein and p-MKP1 infected by SSA1 mutant were much lower than that by WT.Conclusion:SSA1 of C,albicans is related to ite hyphea formation.SSA1 can also induce the expression of a variety of immune effector in oral and vaginal epithelial cells.SSA1 plays a relatively important role for stimulating the differences of certain immune factors in the two types of cells.In the oral cells, SSA1 maybe do not activate the expression of p-MKP1 protein to induce immunity entirely through the ERK immune signaling pathway, while in the vaginal cells, SSA1 induce the expression of p-MKP1 protein through the ERK pathway. SSA1 may be also one of the possible factors that produce the differences of immune in the vaginal epithelial cells and oral cells.Summary:When the oral epithelial cells and vaginal epithelial cells were infected by Candida albicans, The immune response induced in these different sources of epithelial cells is not the same:there are differences in the secretion of inflammatory cytokines and the expression of ERK inflammatory pathway proteins, and this may be one of the mechanisms for the differences in the two cells and two site. By using the cell wall components ALS3 and SSA1 knockout strains of Candida albicans, we found that these strains can reduce the inflammatory cytokine and protein expression in the two epithelial cells, and also eliminated the partial differences between the two cells.So we can conclude the expression of ALS3 and SSA1 genes in Candida albicans has an important role in the induction of immune responses and in the Immunological differences when Candida albicans infects the oral and vaginal epithelial cells. The results of this study can provide more theoretical basis for the prevention and treatment of oral candidiasis and vaginal candidiasis. |
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