Effect Of MB-PDT Against Cariogenic Virulence Factors On Streptococcus Mutans Biofilm

Objective:In this experiment,the photodynamic therapy(Photodynamic Therapy,PDT)mediated by the photosensitizer methylene blue(MB)was used to test the plaque biofilm of Streptococcus mutans(S.mutans)cultured under laboratory conditions.Affect and detect the expression level of caries-related genes of MB-PDT therapy on Streptococcus mutans biofilm.To verify the inhibitory effect of MB-PDT therapy on Streptococcus mutans biofilm,and to explore the sterilization principle and molecular mechanism of MB-PDT therapy by combining real-time fluorescent quantitative PCR to detect the changes of its virulence factors.The application of caries provides a theoretical basis.Method:1.Study on the inhibition of MB-PDT on the growth of Streptococcus mutans biofilm:The Streptococcus mutans plaque biofilm model was established on the cover glass.According to the different treatment methods,it was divided into three groups:(1)MB-PDT treatment group(the concentration of MB was 50μg/ml,The incubation time was 10min;the laser wavelength was 650nm,and the power density was 30.4j/cm~2;(2)the MB concentration was 50μg/ml,the incubation time was 10min;(3)the normal saline control group.According to the experimental methods of each group,the biomembrane model was processed,the living and dead bacteria were stained and observed under the laser confocal microscope.At the same time,the image information was collected.After the image was processed by software,the fluorescence ratio of living bacteria and dead bacteria was calculated,and the living and dead bacteria rate of each group was obtained.2.The effect of MB-PDT on cariogenic factors of Streptococcus mutans According to the same method above,the plaque biofilm model of Streptococcus mutans was established and divided into groups.The samples of each group were treated and cultured for 48h.The biofilm of Streptococcus mutans was collected and the expression of gtf B,gbp A,gbpB,gbpC,gbp D and ffh related genes were detected by real-time fluorescent quantitative PCR.Result:1.Study on the inhibition of MB-PDT on the growth of Streptococcus mutans biofilm:The treated samples were stained with living dead bacteria and observed under laser confocal microscope.It can be seen that there is only a small amount of green signal in the MD-PDT group diagram,and the red signal is strong,indicating that a large number of bacteria died in the biofilm after MB-PDT treatment,which has a good inhibition effect.There are a large area of green signal and less red signal in the group of MB,indicating that there are still a large number of living bacteria in the biofilm after the treatment of MB alone;the image of normal saline group is similar to that of MB group,and the majority of green signal indicates that there are a large number of living bacteria and less dead bacteria in the biofilm in the observation area;Through image processing,the ratio of green signal intensity of each group of samples to the total red green signal intensity is calculated,that is,the proportion of living bacteria.The results showed that the proportion of living bacteria in MD-PDT group was significantly lower than that in normal saline group(p<0.05);there was no significant difference between MB group and normal saline group(p>0.05);the proportion of living bacteria in MB-PDT group was also significantly lower than that in MB group(p<0.05).2.Effect of MB-PDT on cariogenic factors of Streptococcus mutans biofilm:The results of real-time fluorescent quantitative PCR showed that the gene expression of gtf B,gbp A,gbpB,gbpC and gbp D in MB-PDT group was lower than that in normal saline group(p<0.05),and the expression of ffh was not significantly different(p>0.05).There was no significant difference in the gene expression of gtf B,gbp A,gbpB,gbpC and gbp D between MB group and normal saline group(p>0.05),while the gene expression of ffh was slightly higher(p<0.05).The expression of gtf B,gbp A,gbpB and gbpC in MB-PDT group was significantly lower than that in MB group(p<0.05),and there was no significant difference in the expression of gbp D and ffh(p>0.05).Conclusion:Combined with the test results of the biofilm of Streptococcus mutans treated with MB-PDT under the experimental conditions,it can be concluded that MB-PDT has a good sterilization effect on Streptococcus mutans in the biofilm and can significantly inhibit the activity of its biofilm.MB-PDT therapy can reduce the expression of gtf B,gbp A,gbpB,gbpC and gbp D related genes of Streptococcus mutans.It is speculated that the main mechanism of MB-PDT is to reduce the secretion of extracellular matrix of Streptococcus mutans,reduce the synthesis of glucan and destroy the stability of biofilm structure.