| Objectives:To investigate the combination effects of starch,sucrose and high-fructose corn syrup(HFCS)on acid production,adhesive force and the biofilm-forming ability of streptococcus mutans(S.mutans),and further detect the expression of relative cariogenic genes and regulation pathways.Thus we can better understand the cariogenic mechanism of streptococcus mutans and provide a reference for clinical reasonable diet.Material and methods:Part I.Study on the virulence factors of S.mutans S.mutans UA159 were cultured in seven BHI medium containing different types of carbohydrate combination.Four experimental groups are starch+sucrose,starch+HFCS,sucrose+HFCS and starch+sucrose+HFCS;Three control groups are starch,sucrose and HFCS.The p H value of each group was measured at 5 time points and the acid production were compared.The adhesion force of S.mutans was evaluated by crystal violet staining and colorimetry.The morphology of biofilms and the formation of extracellular poly saccharides were observed under laser confocal microscope(CLSM)and the thickness was recorded.Part II.Study on the cariogenic mechanism of S.mutans Real-time PCR was performed to detect the gene expression of extracellular polysaccharide synthesis and degradation,acid production and tolerance,two-component signal transduction system and sugar transport system and explore related gene regulation mechanism.Results:1.The acid production in starch+sucrose group was significantly higher than that in sucrose group after 4,24 and 48h(P<0.05).Apart from the time of 4h when the acid production in starch+HFCS group was significantly higher than that of the HFCS group,the Δp H detected in the former was significantly less than the later at any other time points.And the Δp H in starch+sucrose+HFCS group was significantly higher than the sucrose group at each time point.2.The adhesive ability and the thickness of biofilm formed in starch+sucrose and starch+sucrose+HFCS group were significantly stronger than that of sucrose and sucrose+HFCS group(P<0.05).Adding starch to HFCS,however,lessened the adhesion of S.mutans cultured in HFCS and the biofilm formed in starch+HFCS group showed more polyporous and thinner.3.After 20 h,the expression of gtf B&C and lia S in the group of sucrose+starch were significantly increased(P<0.05)than that of sucrose group,while gbp A,vic R,scr A&B and pts H were decreased sharply.Starch+HFCS significantly increased the expression of gbp C,ldh,atp D,lia S,cia H and pts H when compared with the group of HFCS.The expression of gtf B&C,gbp C,atp F&D and cia R were all up-regulated in starch+sucrose+HFCS group,while the expression of gbp A&D,vic K,scr B and pts I were down-regulated in comparison with sucrose+HFCS.Conclusion:1.The combination of starch and HFCS could up-regulate the expression of ldh gene and therefore promote the acid production of S.mutans.But the interaction between HFCS and sucrose was not obvious.The three kinds of carbohydrate can jointly accelerate the speed and amount of acid production.2.Due to the increased expression of gtf B&C,the adhesive and biofilm-formation ability of S.mutans are significantly enhanced when starch and sucrose are all existed in the environment.However,the addition of HFCS would reduce the thickness of biofilm and inhibit the synthesis of extracelluar polysaccharide.3.The interaction between starch,sucrose and HFCS is related to the precise regulation of the two-component signal transduction system and also affects the sugar transport system of S.mutans.A combination of starch and sucrose could cause changes in the regulatory system of Vis RK and Lia RS,while the interaction between starch and HFCS is mainly involved in the regulation of Lia RS and Cia RH.When starch,sucrose and HFCS all exist in the oral environment at the same time,their interaction can affect the sugar transport system and gene regulation pathway. |
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