| BACKGROUNDRecurrent Oral Ulcers(ROU) also known as "Recurrent Aphthous Ulcers"(RAU) or "Recurrent Aphthous Stomatitis"(RAS),are common lesions of the oral mucosa of which the etiology is unknown.Recurrent Oral ulcers are one of the most common oral mucosal lesions in the general population.The incidence of this pathology can affect 20%of the population.When ROU happens,ROU patients feel severe pain which have bad influence on the ROU patients.There is no significant therapy for ROU.The research on etiology of ROU plays an important role on the therapy for ROU.The etiology of ROU is still unknown.Multifold researches on the etiology show that the generation and development of ROU are related to multiple factors which are studied by many researchers.They refer to immunology,virus,bacteria, microelement,psychology,neurology,genetics and microcirculation.But there is still on consensus on this issue.Inside the human body,there are many microbes,such as oral,intestinal tract, respiratory tract and genitourinary tract.There is an ecological system between these microbes and the human body.This ecological system is a dynamic equilibrium system.There is a commensal relationship between normal flora and the hosts.It is dynamic equilibrium.Because normal flora has double-influence on the hosts,they play an important role on the healthy of the hosts.Usually,the microbes in oral have many advance effect on the hosts,such as to counteract the pathogenic bacteria from outside,to product varied nutrients,to promote immune response,to product superoxide dismutase(SOD),to clean up free radicle(O2-),to resist tumor.The microbes might be harmful to the host,when the balance of this ecological system brokes.Some researchers believe that ROU is a multiple factors process.Multifold researches on the etiology show that the generation and development of ROU are related toveried factors which are studied by many researchers.And there is close relationship between Oral microflora and these factors.This study aims to investigate the variation of Oral microflora in ROU,to determine the correlation between oral microflora and recurrent oral ulcers(ROU),in order to explore some guidelines to the therapy of ROU.MATERIALS AND METHODSâ… ,Observation oral microbial flora through saliva smearCollected salivary samples from 62 candidates who were divided into 3 groups: group T,group C and group N.20 candidates who were ROU patients with oral ulcers were in group T;6 candidates were male and 14 were female,with the mean age on 24.7±3.13 years.21 candidates who were ROU patients with ulcer healing were in group C;9 candidates were male and 12 were female,with the mean age on 25.9±6.67 years.Whereas,21 candidates who were Rou-free individuals were in group N.9 candidates were male and 12 were female,with the mean age on 23.3±2.27 years.All candidates haven't taken any antibiotic,bismuth,antacid and H2 recetor antagonist 1 week before sample collction and steroid,non-steroid anti-inflammatory agent 1 month before.All candidates were without anyother systemic diseases,other oral mocosa diseases and middle or heavy periodontal disease.Methods:saliva samples were smeared directly and do with Gram staining. Observe the bacteria through immersion objective.Choose 5 fields to count the number of G+ coccobacteria(G+c),G- coccobacteria(G-c),G+ bacillus(G+b) and G-bacillus(G- b).Calculate the constituent ratios of G+c,G-c,G+b,G-b.Statistics analysis:the statistical significance of differences in the number of bacteria among the groups was determined by One-way ANOVA.Multiple comparisons were determined by Dunnett T3.All statistical analyses were performed with SPSS 13.0 software.The value of P＜0.05 was defined as significant.â…¡,Quantitation of Streptococcus,Veillonella and NeisseriaCollected salivary samples from 95 candidates who were suitable to the internalize standards.95 candidates divide into 3 groups:group T,group C and group N.39 candidates who were ROU patients with oral ulcers were in group T;13 candidates were male and 26 were female,with the mean age on 26.3±7.76 years.35 candidates who were ROU patients with ulcers healing were in group C;11 candidates were male and 24 were female,with the mean age on 25.3±5.47 years. Whereas,21 candidates who wereROU-free individuals were in group N.9 candidates were male and 12 were female,with the mean age on 23.3±2.27 years.Methods:primers targeted 16S ribosomal RNA gene of Streptococcus sp., Veillonella sp.and Neisseria sp.were designed by Primerprimer 5.0 software. Specificities of primers were verified.Sample bacteria DNA were extracted by CTAB modified method.Quantitations of three common oral microbial populations were detected by real-time PCR.Statistics analysis:the statistical significance of differences in the quantitations of three oral microbial populations among the groups was determined by One-way ANOVA.Multiple comparisons were determined by Dunnett T3.All statistical analyses were performed with SPSS 13.0 software.The value of P＜0.05 was defined as significant.â…¢,Analysis of difference on composition of bacteria in ROUCollected saliva sample from 30 candidates who were suitable to the internalize standards.Candidates divide into 3 groups with 10 candidates in each group:group T,group C and group N.Candidates who were ROU patients with oral ulcers were in group T;candidates who were ROU patients with ulcers healing were in group C; candidates who wereROU-free individuals were in group N.Methods:samples bacteria DNA were extracted by CTAB modified method and made to be pooled samples.Pooled samples 16S ribosomal RNA genes were amplified by polymerase chain reaction,using 16S ribosomal RNA gene generic primers(27f and 1492r).Analysis of difference on composition of bacteria was performed by agarose gel electrophoresis and AlphaEaseFC Stand Alone softwera. Several significant different fragments were purified,and sequenced and matched to the GenBank database.RESULTSâ… ,Observation oral microbial flora through saliva smearThe constituent ratio of G+c in group T,C and N was:45.16±6.99,43.38±6.92 and 42.96±4.69;the constituent ratio of G+b was:3.95±3.12,3.04±1.38 and 2.65±1.71;the constituent ratio of G-b was:4.94±2.43,3.79±2.16 and 4.35±2.96. There are no statistically significances on the constituent ratios of G+c,G+b,G-b among group T,C and N(P=0.499,P=0.160,P=0.352).The constituent ratio of G-c in three groups was:45.94±5.42,49.79±6.22 and 50.09±4.65;There is statistically significance on the constituent ratio of G-c.The constituent ratio of G-c in group T was 3.85 points lower than group C and 4.15 points lower than group N. The constituent ratio of coccobacteria in saliva microbial flora was above 90%.The constituent ratio of coccobacteria in three group was:91.10±4.83,93.17±2.57, 93.05±4.38:the constituent ratio of bacillus in three group was:8.89±4.83, 6.83±2.57,7.00±4.37;there are no statistically significances on the constituent ratio of coccobacteria and bacillus among group T,C and N(P=0.193,P=0.201).The ratio of G+c/Gc in group T,C and N was:1.00±0.26,0.90±0.26,0.87±0.16;the ratio of G+b/G-b was:0.83±0.45,1.13±1.01,0.64±0.30;the ratio of G+c/G+bwas: 17.51±13.55,18.08±11.02,24.20±15.93;the ratio of G-c/G-bwas:12.23±7.14, 17.77±12.10,15.34±7.90.There are no statistically significances on the ratios of G+c/G-c,G+c/G+b,G-c/Gb among group T,C and N(P=0.140;P=0.063,P=0.224, P=0.172).The ratio of G+b/G-b in three groups was different,P=0.063.The constituent ratio of G+c/(G+c+ G+b) in group T,C and N was:0.92±0.07,0.93±0.03, 0.94±0.04;G+b/(G+c+ G+b):0.08±0.07,0.07±0.03,0.06±0.04;G-c/(G-c+ G-b): 0.90±0.04,0.93±0.04,0.92±0.05;G-b/(G-c+ G-b):0.10±0.04,0.07±0.04, 0.08±0.05;G+c/(G+c+ G-c):0.49±0.06,0.47±0.07,0.46±0.04;G-c/(G+c+ G-c): 0.51±0.06,0.53±0.07,0.54±0.04;G+b/(G+b+ G-b):0.43±0.11,0.46±0.18,0.37±0.10; G-b/(G+b+G-b):0.57±0.11,0.54±0.18,0.63±0.10.There are no statistically significances on the constituent ratios of G+c/(G+c+G+b),G+b/(G+c+ G+b), G-c/(G-c+G-b),G-b/(G-c+ G-b),G+c/(G+c+ G-c),G-c/(G+c+ G-c),G+b/(G+b+ G-b),G-b/(G+b+ G-b)(P=0.302,P=0.302,P=0.162,P=0.162,P=0.166,P=0.166, P=0.117,P=0.117). â…¡,Quantitation of Streptococcus,Veillonella and NeisseriaThe logarithms of the number of Streptococcus sp.and Veillonella sp.in group T are significant lower than group N(7.30±0.89 vs 8.15±0.55 and 8.29±0.77 vs 8.93±0.76,P＜0.01).There are no statistically significances on the logarithms of the number of Streptococcus sp.and Veillonella sp.between group T and C(P=0.64; P=0.17).The logarithm of the number of Streptococcus sp.in group C is significant lower than group N(7.51±0.81 vs 8.15±0.55,P＜0.01).There is no statistically significances on the logarithm of the number of Veillonella sp.between group C and C(P=0.056).There is no statistically significance on the logarithm of the number of Neisseria sp.among group T,C and N(P=0.921).â…¢,Analysis of difference on composition of bacteria in ROUThere were 3 stable straps which was on 230bp,400bp and 770bp were recognized by AlphaEaseFC Stand Alone in group T,C and N between 200～800bp. The gradation of 3 stable straps was different among three groups.The constructions of all recognized straps in three groups were different.Some straps were absence in group T and C.Reclaim and purify one fragment from agarose gel and name them as 1.The length of 1 is 180bp.Sequencing and bioinformation analyses were operated. Blast to the GenBank,1 is 70%isogenic to the Bacteroides vulqatus ATCC 8482(ID: CP000139.1),with 74%matching.CONCLUSIONâ… ,The results of saliva smear indicates that the main saliva microbial flora is coccobacteria.And the constituent ratio of G-c in group T was lower.It indicated that the construction of oral flora changed in ROU.â…¡,The contents of Streptococcus sp.and Veillonella sp.in saliva from ROU reduced.It indicated that the content of important oral microbal populations changed in ROU.â…¢,Analysis of 16S rRNA gene prouducts by agarose gel electrophoresis and AlphaEaseFC Stand Alone indicated that the construction of oral bacteria was different between ROU and ROU-free individuals.On the whole,the content and the construction of oral microbal population in ROU have changed.It indicated that the occurrence and development of ROU were related with oral microecology.Doctors shoud pay attention to the adjustment of oral micrioecological system in the therapy of ROU.
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