| As we know, dental caries is a chronic infectious disease in dental hard tissues with ahigh incidence. Along with the bacteria gathering, adhering, metabolism, acid producing, the tooth got partial demineralization and ultimately developed caries. Streptococcus mutans (S mutans) is recognized as the major etiological bacteria. It has adapted to the biofilm lifestyle, which is essential for pathogenesis of dental caries. On the basis of physical reversible attachment to the tooth surface, those bacteria consolidated themselves via virulence factors on the surface, and construct the cariogenic biofilm at last. The biofilm provides a relatively stable niche for the bacteria inside which lead to a higher resistance. So the study on the effect of cariogenic Streptococcus mutans biofilm has important significance in caries prevention.Previous studies for Streptococcus mutans biofilm mainly focused on antibiotics. But with the increase in clinical drug-resistant strains, people gradually develop awareness of the ecological balance. In this case, scholars start to look for natural compounds for bacterial inhibition. In recent years, marine natural product is a rich source of novel chemicals for the discovery of more effective drugs. Small molecules of marine product have strong biological activity and have been shown to inhibit a variety of Gram-negative bacteria as well as a small amount of Gram-positive bacteria. In our study, we research on a novel small marine biological molecule2-amino-imidazole/triazole (2-AI/T) to investigate its effect on Streptococcus mutans biofilm, as well as the toxic effects of drug use, and initially explore its anti-biofilm mechanisms.Our study consists four parts:Part oneMethods:Firstly, small molecule2-AI/T was double diluted and obtained a low concentration gradient of the drug. Streptococcus mutans biofilm co-cultured with the drug for16h, then the inhibition effect of the drug was tested by crystal violet staining. IC50values was calculated according to the result; In the mean time, after drug intervention biofilm formation was observed with confocal microscopy (CLSM). Secondly, we test biological activity of both planktonic bacteria and biofilm in consecutive24hour respectively using MTT method and CFU counting. Thirdly, we prepared the high concentration gradient of small molecule drugs in order to determine its clearance on the newborn S mutans biofilm in4h via adherence testing. Result: in the inhibition test, along with the augment of2-AI/T concentration, the effect of inhibition increase, and the IC50equals to75umol/1. In the bioactivity test, we found a continuing inhibitive effect of2-AI/T towards S mutans in biofilm, which increases with the time passing; While the obvious inhibitive effect on floating bacteria only lasts for1Oh, and after that it wears off. In the clearance tests, we demonstrate that2-AI/T greater than125umol/1have pellant effect on new born biofilm. Conclusion:the in-vitro study shows that the compound2-AI/T have a IC50equals to75umol/1, which has an obvious inhibit effect.2-AI/T could significantly inhibit the UA159biofilm as well as planktonic cell and the inhibited time for the former one is longer. Besides,2-AI/T could clear the biofim in the initiate stage.Part twoMethods:In the first place, a low concentration gradient of small molecule2-AI/T was prepared, and then co-culture with human gingival fibroblasts. The toxicity level was calculated by MTT method. In the second place, inorder to form acute toxicity model,20mice was intragastric administrated with2-AI/T in large dose. After14days, mice was weighed and its blood or urine was taken for biochemical identification. Then sacrifice mice respectively taking heart, liver, spleen, kideny and lung for histological assay, result: The growth activity gingival fibroblasts have no differences between group2-AI/T and control. Both biochemical identification and histological assay showed group using2-AI/T have no difference with control group. conclusion:2-AI/T less than100umol/1have no toxicity on gingival fibroblasts. Animal model showed2-AI/T have no acute toxic risk. Part threeMethod:In the biofilm model. BABL/C were fed with cariogenic diet and orally administrated with S.mutans or2-AI/T. in order to confirm if2-AI/T still have inhibitive effect in vivo, after1,3,5days, the infection rate of S.mutans in tooth surface was taken by cotton pledget and calculated by colony forming unit counting. In the caries model, pre-treatment of SD rat was similar with BABL/C, but the dose of2-AI/T or S.mutans was different. After all rats suffered for70days, they were sacrifice for tooth checking. Micro-CT was used for observing the tooth and calculated the volume of defect, in order to identify the anti-caries effect of2-AI/T. Result: after bacteria successfully colonization, the2-AI/T have obvious inhibitive effect towards S.mutans in vivo for at least5days. Mcro-CT showed an obvious larger defect in control group which was statistically significant. Conlusion:2-AI/T showed a anti-caries effect in vivo which made it a potential drug in caries.frevetion.Part fourMethod:A novel technology was used in this part, which called iTRAQ(isobaric tag for relative and absolute quantitation). Simply describe the processes into four part: extracted protein from bacteria, split protein into small peptides, labeled by a fluorescent peptide. analyzed specimen source through computer to calculate the difference between drug treated groups and the control group. Result: a total of394points were showed difference with statistically significant, including up16, down15.Conlusion:2-AI/Thas a functional effect on Streptococcus mutans which leads us to an in-depth research. |
PDF Full Text Request