The Relationship Between The Formation Of Biofilm Implanted With Candida Albicans - Staphylococcus Epidermidis Biofilm And The Immune Function Of The Host

The part I:Candida albicans-staphylococcus epidermidis mixed specices biofilm in vitro model and structure observation[Objective]:To establish an in vitro model of Candida albicans-Staphylococcus epidermidis mixed species biofilm, and to investigate mixed species biofilm formation and its microstructure. Evaluate Candida albicans-Staphylococcus epidermidis mixed species biofilm formation in different biomaterials surface.[Methods]:Staphylococcus epidermidis bacteria (ATCC35984) and Staphylococcus epidermidis bacteria (ATCC12228) co-incubated with Candida albicans fungal (ATCC10231) respectively on 0.5 cm diameter PVC pieces in Tris buffered saline to form mixed specie biofilms. Bio-film formation was examined at2,6,12,24,48, and 72 h.Thicknesses of these biofilms and the number, and per-centage of viable cells in biofilms were measured. CT scan images of biofilms were obtained using CLSM and SEM. Reconstruct 3D mixed specice biofilm images.Using PVC material cultured in the TSB medium as control group, co-incubated Staphylococcus epidermidis bacteria (ATCC35984) with Candida albicans fungal (ATCC 10231) in PVC, silicone rubber, polyurethane material respectively, Bio-film formation was examined at2,6,12,24, 48, and 72 h.Thicknesses of these biofilms and the number, and per-centage of viable cells in biofilms were measured by CLSM.[Result]:CLSM shows colonies and biofilm formation can be found after 6h co culture, and gradually increasing with the time. pathogenic bacteria colonies reached the peak at 24h,and biofilm maximal thickness being attained at 48h, biofilm thickness trend stable at 72h;The biofilm thickness in Ca-Se+ group at 6h、12h、24h、 48h and 72h were higher than Ca-Se- group.The number of colony of Ca-Se+ group were significantly more than Ca-Se- group among 6h、12h、24h、48h、72h time piont(P <0.05),and no significantly different were found among 24、48、72h (P>0.05).As CLSM and 3D reconstruction imge displayed:the surface of mixd sepecies is uneven, Protrusive places were mainly living bacterium,dead bacteria mainly locate in concaves.SEM image showed with the increased of occulted time, Staphylococcus epidermidis attached to various forms of candida albicans (spores, pseudohyphae, hyphae) gradually, formed dense array of mesh stereochemical sophisticated structure on the surface of PVC. Compared with Ca- Se- group, in the Ca-Se+ group biofilm structure is more complex, candida albicans and staphylococcus epidermis stick closer, with more staphylococcus epidermis candida albicans adhesion.The result of CLSM test show the Ca-Se+ biofilm thickness were significantly different in PVC、silicone rubber and polyurethane, silicone rubber is greatest, PVC followed and polyurethane was the lowest.[Conclusion]:Candida albicans-Staphylococcus epidermidis mixed sepceie biofilm is sophisticated in structure, The Ca-Se+ group formed more complex structure and thickness mixed specices biofilm than Ca-Se- group.The ability of Candida albicans-Staphylococcus epidermidis mixed sepceie biofilm formation on silicone rubber is strongest, PVC followed and polyurethane is the lowest. concaves.SEM image showed with the increased of occulted time, Staphylococcus epidermidis attached to various forms of candida albicans (spores, pseudohyphae, hyphae) gradually, formed dense array of mesh stereochemical sophisticated structure on the surface of PVC. Compared with Ca- Se- group, in the Ca-Se+ group biofilm structure is more complex, candida albicans and staphylococcus epidermis stick closer, with more staphylococcus epidermis candida albicans adhesion.The result of CLSM test show the Ca-Se+ biofilm thickness were significantly different in PVC、silicone rubber and polyurethane, silicone rubber is greatest, PVC followed and polyurethane was the lowest.The part Ⅱ:The function expression of Candida albicans Efgl, Ecel, Sap5 gene and staphylococcus epidermis AltE, Aap gene in the formation of biomaterial-related candida albicans-staphylococcus epidermidis mixed specice biofilm in lung cancer patients[Objective]:To explore the function of Candida albicans Efgl, Ecel, Sap5 gene and the staphylococcus epidermis AltE, Aap in the process of Ca-Se mixed specice biofilm formation of lung cancer patients.[Methods]:Co-cultured staphylococcus epidermidis strains ATCC35984 (Se+) and candida albicans strains ATCC10231 (Ca) polyvinyl chloride (PVC), add advanced non-small cell lung cancer patients and healthy people serum respectively, divided to the lung cancer group and healthy group, No added as control gourp. At cultured 2,6, 12,24,48,72 h time point, CLSM test the biofilm thickness and the colony number on the PVC surface of two gourps; and in co-culture 6 h,24 h,48 h, fluorescence quantitative PCR to detect candida albicans Efgl, Ecel, Sap5 gene and the staphylococcus epidermis AltE, Aap gene expression differences on the PVC material of each groups.[Result]:CLSM show:at 6 h,12 h culture in lung cancer group and healthy group of PVC material surface colony count more than control group, lung cancer group of PVC material surface colony count more than the healthy group (p< 0.05); Trained 12 h,24 h,48 h when lung cancer hybrid biofilm thickness is greater than the healthy group and the control group, the difference is statistically significant (p< 0.05). Fluorescence quantitative PCR, according to the results of three groups of AltE cut obviously with the increase of total training time, statistically significant difference (P < 0.05),6 h AltE express the lowest lung cancer group, healthy people group, control group is highest, comparing two differences statistically significant (P< 0.01); Aap expression in three groups raised significantly with the increase of total training time, 6 h control Aap expression in the control group, the second lung cancer group, highest lowest health group, comparing two differences statistically significant (P< 0.05); 12 h Aap second highest expression in lung cancer group and healthy group and the control group, two two compared statistically significant difference (P< 0.05); Three groups of Efg1 cut obviously with the increase of total training time, statistically significant difference (P< 0.05),6 h Efgl express the highest lung cancer group, healthy people group, control group, comparing two differences statistically significant (P< 0.01),12 h health Efgl expression in lung cancer group and healthy group and control group, there was no statistically significant difference comparing the two (P> 0.05),48 h Efg1 expression in lung cancer group and health has no statistical significance (P> 0.05), but lower than the control group (P> 0.05). Ecel expression in three groups raised significantly with the increase of total training time, 6 h control Ecel second highest expression control function and lung cancer group and healthy group, the lowest two comparative differences statistically significant (P < 0.05),24 h, the lung cancer group and control group Ecel expression is lower than the healthy group, difference is statistically significant, there was no statistically significant difference between lung cancer groups and control group (P> 0.05). Sap5 expression in three groups increases obviously with the increase of total training time, 6 h three groups of Sap5 expression in the control group, the second lung cancer group, highest lowest health group, the two comparative differences are statistically significant (P< 0.05); 48 h, Sap5 highest expression in lung cancer group and healthy group, control group, the lowest two comparative difference was statistically significant (P< 0.05).[Conclusion]:Compared with the healthy crowd epidermis staphylococcus and mixed cultivation in c. albicans in lung cancer patients implanted biomaterials more surface mixed biofilm formation, early may and cultivate candida albicans Efg1 cut, Ecel, Sap5 gene expression quantity expression quantity increases and the epidermis staphylococcus AltE, Aap cut the amount of gene expression. and healthy group, the lowest two comparative differences statistically significant (P < 0.05),24 h, the lung cancer group and control group Ecel expression is lower than the healthy group, difference is statistically significant, there was no statistically significant difference between lung cancer groups and control group (P> 0.05). Sap5 expression in three groups increases obviously with the increase of total training time, 6 h three groups of Sap5 expression in the control group, the second lung cancer group, highest lowest health group, the two comparative differences are statistically significant (P< 0.05); 48 h, Sap5 highest expression in lung cancer group and healthy group, control group, the lowest two comparative difference was statistically significant (P< 0.05).The part III:The effects of lung cancer biomaterial implant candida albicans-mixed by staphylococcus epidermidis biofilm on cellular immunity[Objective]:Comparison of lung cancer patients and healthy people peripheral blood mononuclear cells (PBMCs) on candida albicans-epidermis staphylococcus biofilm, TLR2 and TLR4, TLR9 receptor expression, T lymphocyte differentiation and the related changes of cytokine secretion.[Methods]:Density gradient centrifugation separated peripheral blood mononuclear cells of advanced lung cancer patients and healthy people in 10 cases. Co-cultured staphylococcus epidermidis strains ATCC35984(Se+) and candida albicans strains ATCC10231 (Ca) polyvinyl chloride (PVC) 48h. Separation of supernatant on mixed species biofilm. Supernatant cultured with PBMCs in different groups.RT-PCR detect the express change of TLR2、TLR4、TLR9. Flow cytometry to detect the concentrate change of IL-2、IL-4、IL-6、IL-10、TNF and INF-gamma, and count CD3+、CD4+、 CD8+、CD3+CD25+、CD4+ CD25+cell proportion.[Result]:(1) RT-PCR result show in lung cancer group than PBMCs cells TLR2 and TLR4 expression is lower than the healthy group (P< 0.05), TLR9 has no difference between the two groups (P> 0.05). With candida albicans-epidermis staphylococcus aureus biofilm trained after 24 h, after 24 h cultured with candida albicans-staphylococcus epidermis mixed biofilm supernatant, PBMCs TLR2、TLR4、TLR9 expression in lung cancer group and healthy group under the lung cancer group was higher than healthy group (P< 0.05). (2)Flow cytometry to detect show lung cancer group and healthy group with control group Th1 factor:IL-2 and TNF, IFN-gamma density decreased significantly (P< 0.05), the Th2:IL-4, IL-10 rise significantly (P < 0.05), degree of Th1 factor lower lung cancer group is higher than healthy group (P < 0.05), and the affecting factors of Th2 raised degree level higher than the healthy group (P< 0.05);(3) Trained before lung cancer group compared with the healthy group of CD3+, CD4+, CD8+ cell ratio is lower than the healthy group, CD4+ CD25+ cells percentage is higher than the healthy group; Trained after 24 hours compared with control group, compared with the control group two groups of CD3+, CD4+ cell ratio decreased significantly (p< 0.05), CD8+, CD4+CD25+cells increased significantly (p< 0.05); Lung cancer group of CD3+, CD4+, degree of cell percentage is less than the healthy group (p< 0.05), lung group of CD8+, CD4+ CD25+cells percentage rising degree is less than the healthy group (p< 0.05).[Conclusion]:Candida albicans and staphylococcus epidermis hybrid biofilm supernatant can cut in human peripheral blood mononuclear cells on the expression of TLR2 and TLR4 and TLR9, lead to the cellular immune response toward Th2 type of immune response development, leading to the quantity of cytokines, it may be lung cancer patients with BF relatively healthy people easy to escape the body’s immune defense system.