HGFs Were Influenced And IL-1β,IL-6,MMP-2 Activated By The Supernatant Of Pg,Pi,Aa And Fn

Postgraduate:Huang DiAdvisor:AssoeiateProfessor Shui yan qingHGFs were influenced and IL-1β,IL-6,MMP-2 activated by the supernatant of Pg,Pi,Aa and FnObjeetive:This study investigated the effects of supernatant of Pg,Pi,Aa and Fn of different concentration gradient influenced human gingival fibroblasts (HGFs) in vitro. The inflammatory cytokinegene expression was observed in human gingival fibroblasts stimulated with supernatant of Pg,Pi,Aa and Fn.Methods:HGFs were cultured in vitro using the primary tissue explant method. The 6 passages were used for the experiment. The cells were seeded onto 96-well at the concentration of 2x 104 cells/well, and cultured for 24h. Then they had been stimulated cells with supernatant of the four anaerobic bacterium concentrations of 1×108 CFU/mL,1×107 CFU/mL,1×106 CFU/mL,1×105 CFU/mL,1×104 CFU/mL,1×103 CFU/mL and 1×102 CFU/mL; the control was deal with of Dulbecco's modified Eagle's (DMEM) containing 1% fetal bovine serum (FBS). At 24h and 48h, the cells multiplication were assayed by means of Methyl thiazolyl tetrazolium(MTT); Stimulated 2×105 cells/mL with supernatant of the four anaerobic bacterium concentrations of 1×106 CFU/mL for 48h, assayed for cytokines in the culture supernatants employed commercially available Enzyme-Linked Immnosorbent Assay (ELISA) kits obtained from the following sources:Interleukin-1βand Interleukin-6; Assayed for Interleukin-1β, Interleukin-6 as well as MMP-2 expression by means of RT-PCR. All assays were conducted in accordance with the manufacturer's instructions.Finally used one-way ANOVA and factorial design analyze ANOVE to analyze the date.Result:At 24h, contrast with control group, the superment of Pg,Pi,Aa and Fn at the concentrations of 1×108 CFU/mL,1×107 CFU/mL,1×106 CFU/mL,1×105 CFU/mL,1×104 CFU/mL,1×103 CFU/mL and 1×102 could inhibit HGFs, and the data was significant (P<0.05). At 28h, contrast with control group and 24h group, the superment of four anaerobic bacterium at the concentrations of 1×108 CFU/mL 1×107 CFU/mL,1×106 CFU/mL,1×105 CFU/mL,1×104 CFU/mL,1×103 CFU/mL and 1×102CFU/mL could inhibit HGFs, and the data was significant (P<0.05), and significantly more effective than 24 group;The outcome of RT-PCR shows that there express IL-6 and MMP-2, except IL-1β;The outcome of ELISA shows that the production of IL-6 much more than IL-1β(P<0.05)Conclusion:1,At 24h, supernatant of Pg,Pi,Aa and Fn at the concentration of 1×108 CFU/mL,1×107 CFU/mL,1×106 CFU/mL,1×105 CFU/mL,1×104 CFU/mL,1×103 CFU/mL and 1×102 CFU/mL were able to inhibit HGPs proliferation;2,At 48h, supernatant of Pg,Pi,Aa and Fn at the concentration of 1×108 CFU/mL,1×107 CFU/mL,1×106 CFU/mL,1×105 CFU/mL,1×104 CFU/mL,1×103 CFU/mL and 1×102 CFU/mL could inhibit HGPs proliferation, and significantly more effective than 24 group;3,Supernatant of the four anaerobic bacterium inhibition HGPs proliferation were time-and- concentration dependence:4,The work of inhibition from best to bad was Pg,Pi,Aa and Fn;5,At 48h, supernatant of the four anaerobic bacterium at the concentration of 1×106 CFU/mL stimulated cells, the outcome of RT-PCR shows that there express IL-6 and MMP-2, except IL-1β; and the outcome of ELISA shows that the production of IL-6 much more than IL-1β(P<0.05)...