|
Plaque biofilm (Dental plaque biofilm) is the initiating factor of caries, cariogenic bacteria in plaque through multiple virulence factors in the incidence of dental caries process played an important role, mainly in adhesion, acid and acid three links. Cariogenic bacteria which acid is the most important biological characteristics of dental caries is also the premise that only can tolerate acidic environment to continue cariogenic bacteria metabolize and produce acid, resulting in occurrence of enamel demineralization caused by caries. Membrane proton translocating ATP enzyme (membrane-bound proton-translocating ATPase, F0F1ATPase, F-ATPase, H-ATPase, F-type ATPsae) transit through the extracellular protons in order to maintain a relatively neutral intracellular environment, thereby protecting cells the acid-sensitive enzymes and other proteins that have the right antacid cariogenic bacteria lethal capabilities and survive at low pH conditions continue to produce acid, so as to achieve the critical pH of enamel demineralization, therefore, F-ATPase is cariogenic The most important acid bacterial virulence factor. Dental plaque is a biofilm in situ cariogenic bacteria pathogenic environment, it is not a simple sum of bacterial structure, but built up a complex microbial community, with biofilm structure and function of microbial physiology, bacterial biofilms exist between mutual antagonism, interdependence and signal transduction, start a completely different genetic systems planktonic affect cariogenic bacteria virulence factor expression and toxicological effect, it is observed biofilm F-ATPase expression can accurately reflect the true cause caries acid bacteria virulence factors in the development of dental caries in rats. Objective: This topic detection Acid Factor F-ATPase in a multi-bacterial artificial oral biofilm (in vitro biofilm, artificial biofilm, in vitro biofilm) and clinical caries susceptible individuals of different gene expression in situ plaque characteristics and enzyme activity, and observed F-ATPase promoter luciferase reporter gene plasmid was transformed bacteria biofilms at different pH environments fluorescence expression, a preliminary analysis of virulence factors F-ATPase in biofilms gene and protein expression levels, to further clarify the caries etiology, disease risk assessment and prevention are important. Methods: a quantitative PCR artificial oral biofilm F-ATPase at different pH conditions, the difference in the mRNA expression of Streptococcus mutans (S. mutans), Streptococcus sanguis (S.sanguis), Streptococcus sobrinus (S . sobrinus), within Actinomyces (A. naeslundii), Actinomyces (A. viscosus), oral streptococci (S.oralis) and Neisseria (Neisseria) 8 种 build oral bacteria associated with dental caries In vitro biofilm model, different pH environments grow 3h, biofilm samples extracted RNA, reverse transcriptase cDNA, fluorescence quantitative PCR (SYBR Green I) detects F-ATPase mRNA expression. ANOVA statistical results. Second, quantitative PCR different caries susceptible individuals plaque biofilm F-ATPase mRNA expression were collected from adult caries (61 cases) and without caries (50 cases), the elderly root surface caries (56 cases) and root surface caries (41 cases) populations plaque, plaque extracted RNA, reverse transcriptase cDNA, fluorescence quantitative PCR (SYBR Green Ⅰ) detect plaque F-ATPase mRNA expression. ANOVA statistical results. Third, artificial oral biofilm F-ATPase enzyme activity of the same expression experiments a constructed artificial oral biofilm permeability preparing mixed bacterial biofilm cells under different pH conditions to detect phosphorus release to reflect the F-ATPase activity . ANOVA statistical results. Four, pre-built F-ATPase promoter transformed bacteria growth and recombinant expression vectors fluorescent artificial oral biofilm in the fluorescence characteristics of subjects expressing pre-built F-ATPase promoter shuttle vector PLFgfp green fluorescent protein and red fluorescent protein housekeeping gene recA shuttle vector PLRred, electroporation of plasmid DNA Streptococcus mutans (UA159), the effects of different pH conditions grown bacteria BHI agar containing kanamycin plates of colony growth, the laser scanning confocal microscopy two kinds of shuttle vector in E. coli under different pH conditions fluorescence biofilm expression Results: 1. Artificial oral biofilm F-ATPase expression at different pH conditions are different, the initial pH 5 and 5.5 when the F-ATPase was the highest, followed by the initial pH 6,6.5,4.5,7, the initial pH 4 When was the lowest. Acidic environment is relatively neutral environment (pH 7) F-ATPase expression differences were statistically significant (P lt; 0.05). (2) high caries group of people F-ATPase was significantly higher than those without caries group (P lt; 0.01), high caries group adult F-ATPase was higher than the elderly (P lt; 0.01), with no caries group in adults and the elderly were no significant differences (P gt; 0.05). F-ATPase in situ in individual clinical expression of plaque biofilm is much higher than artificial oral biofilm (P lt; 0.01). 3 in the initial stage acid treatment prior to 10min, F-ATPase activity decreased with increasing pH; after 3h incubation stability, pH 5 and 5.5, the highest activity, followed by a pH 6, pH 4.5, pH 6.5, pH 4, the weakest activity at pH 7. Relatively neutral environment (pH 7) acidic environment F-ATPase activity of the differences were statistically significant (P lt; 0.05). 4. F-ATPase promoter plasmid after electroporation of Streptococcus mutans can be grown in BHI containing kanamycin plates, PCR amplification results confirm successful conversion, at pH 5.5 transformed bacteria kanamycin resistance plate the highest growth rate, and then followed by pH 5, pH 6, pH 6.5, pH 7, pH 4.5, the growth rate is the lowest pH 4, pH after culture results are similar to the multiple different bacterial artificial oral biofilm F-ATPase expression . F-ATPase promoter and an internal reference recA promoter recombinant plasmid was transformed into E. coli after the same experimental conditions to build an artificial oral biofilm different pH, confocal laser scanning microscope fluorescence expression, the results show: green fluorescence (F-ATPase start Sub recombinant plasmid) at an initial pH of 5.5 expressed in most conditions, followed by the initial pH 5, pH 6, pH 4.5, pH 6.5, pH 4 and pH 7, and the red fluorescence (internal reference recA promoter plasmid) of Expression of the initial maximum at pH 7, with the decrease of pH reduction in the initial pH 4, expressed less. Conclusion: the biofilm, pH value affects F-ATPase gene expression and activity of the initial pH 5-5.5 environment both gene expression and enzyme activity highest Streptococcus mutans F-ATPase promoter fluorescent protein environment at the initial pH 5.5 expression of the strongest (pH 5-5.5 is enamel demineralization and remineralization imbalance caused by caries critical pH); crowd situ caries plaque F-ATPase expression is much higher than those without caries group, found bacteria cariogenic plaque biofilm microorganisms through F-ATPase gene expression and protein expression increased at low pH environment of enhanced acid tolerance and cariogenic.
|