Study On The Expression And The Patterns Of DNA Methylation Of Occludin In Infective Inflammatory Mucosal Epithelium

PART1Establishment of infective inflammation mucousepithelium in rats modelsObjective: To establish infective inflammation mucous epithelium ratsmodel with/without diabetes, and create foundations for the furtherstudy on immune mechanism of Occludin protein in infectiveinflammation mucous epithelium.Method:60male Wistar rats were randomly divided into normalcontrol group (group N), diabetes mellitus group (group D),periodontitis group (group P) and diabetic periodontitis group (groupDP) of four groups. Single intravenous injection of streptozotocin(Streptozotocin, STZ) method was used to induce diabetic rat model in Group D and group DP; Group P and group DP were treated by tiringsilk thread to teeth combined with periodontal inoculation ofPorphyromonas gingivalis (Porphyromonas gingivalis, P.g), givinghigh sugar diet in the meantime. Maxillary first molar and gingivalepithelial tissue were taken after4weeks, fixed with4%paraformaldehyde, embedded in paraffin, Histopathologicalexamination were conducted to comfirm the success of animal model.situation of gingival epithelial and inflammatory classification of ratsin each group were compared.Results: Clinical symptoms of diabetes,such as Polydipsia, polyphagia,polyuria, weight loss, easy to infection were observed in diabetic rats.Blood glucose of group D and DP was higher than that of group N（P<0.05）.In group N, HE staining results showed normal periodontalsoft tissue and periodontal attachment, no inflammatory cell infiltration.In group D, a small amount of inflammatory cell infiltration could beobserved in periodontal soft tissue, no significant periodontal pocketwere found. In group P, obvious inflammatory cell infiltration could befound in periodontal soft tissue. In group DP, the most seriousperiodontitis gingival epithelial inflammation could be found, significant difference could be found when compared with group P(P<0.05).Conclusion: Infective inflammation mucous epithelium rats modelwith/without diabetes were successfully established. Diabeticperiodontitis rats suffer the most serious eriodontitis gingival epithelialinflammation. PART2Expression of occludin in infective inflammatorymucosal epithelialObjective: To detect the expression and distribution of occludin, a kindof tight junction protein of epithelial, in infective inflammation mucousepithelium rats model with/without diabetes. Methods: Immunohistochemical methods were used to detect theexpression and distribution of occludin in infective inflammationmucous epithelium rats model with/without diabetes. RT-PCRsemi-quantitative detection were used to observe the mRNAexpression of occluding protein. SPSS16.0statistical analysis wasused to analyze the data.Results: Occludin protein positive staining mainly located on themembrane and cytoplasm in superficial cells with brown color. Innormal gingival mucosa, the expression of occludin is compact andcontinuous with uniform coloring. In diabetes mellitus group,periodontitis group and diabetic periodontitis group, the expression ofoccludin were interrupted and disorder, especially in diabeticperiodontitis group, loss of occluding could be found. Expression ofoccludin mRNA in periodontitis group and diabetic periodontitis groupwere decreased compared to that of normal control group, thedifference was statistically significant (P<0.05); occludin mRNAexpression in diabetic periodontitis group is lower than that ofperiodontitis group, statistically significant difference could befound(P<0.05). Conclusion: Diabetes and periodontitis rats are more susceptible toperiodontal mucosal barrier injury, especially in diabetic rats.Decrease of occludin expression and diabetes may promote theprocess of infective inflammatory gingival mucosa injury, result in theinvasion of pathogenic microorganism, further cause the destructionof the gingival mucosa barrier. These results may provide basis for theprevention and treatment of periodontal mucosal injury. PART3Study on occludin DNA methylation in infectiveinflammatory mucosal epithelialObjective: Understanding of the infective inflammatory mucosalepithelial occludin (occludin) DNA methylation status, and its effect onthe mucosal epithelial barrier. Methods: Methylation specific PCR (MSP) were used to dectectoccludin (occludin) DNA methylation. SPSS16.0statistical analysiswas used to analyze the data.Results: The occludin gene promoter methylation status were detected,all specimens after bisulfite treatment had occludin DNAnon-methylatetd product, occludin DNA methylation products were notfound. The number of cases of occluding DNA methylation andmethylation rate were0.Conclusion: Although occludin expressed in gingival mucosa tissue,but occludin DNA methylation may have not the exact relationshipwith the occurrence and the severity of gingival inflammation.