| Objective In this experiment,established mice models of periodontitis by applying porphyromonas gingivalis(P.gingivalis)W83 to the oral cavity of mice.We used intraperitoneal injection of anti-GITR m Ab to block Treg cell function.To observe the effect of Treg cells on the immune response mediated by type M1 macrophages in periodontitis mice.Methods1.Twenty female 7-week-old SPF C57 BL / 6 female mice were randomly divided into 4 groups,5 in each group: normal control group,periodontitis group,Ig G group and anti-GITR m Ab group.2.The mice periodontitis models were established by P.gingivalis W83 continuous oral smear for 7 days.Anti-GITR m Ab mice received P.gingivalis W83 on the first day of oral application.Anti-GITR m Ab(100 μg / mice)was injected intraperitoneally every 3 days for a total of 4 times.Mice in the Ig G group were injected intraperitoneally with Ig G isotype control antibody(100 μg /mice).3.The body weight changes of the 4 groups of mice were dynamically monitored,and samples were taken on the 45 th day after infection.4.The gingival index and tooth mobility of the mice were observed under a stereo microscope.5.The distance between the cemento-enamel junction(CEJ)and the alveolar bone crest(ABC)was measured to evaluate the alveolar bone absorption under a stereo microscope.6.Hematoxylin and eosin(HE)staining was used to observe the periodontal tissue infiltration of inflammatory cells,loss of connective tissue attachment and alveolar bone resorption.7.Real-time quantitative RT-PCR was used to detect the relative expression levels of IL-10,TGF-β1,Foxp3 and i NOS m RNA.Results1.Compared with the normal control group,the weight of mice in the D14 pure periodontitis group,the Ig G group and the anti-GITR m Ab group began to decrease.And there was no significant difference in the weight loss of the mice with pure periodontitis group and Ig G group(P > 0.05).The weight loss of mice in the anti-GITR m Ab group was significantly different(P <0.05).Compared with the normal control group,there was a significant difference in weight loss in the D35 between pure periodontitis group,Ig G group and anti-GITR m Ab group(P<0.01).Compared with pure periodontitis group and Ig G group,the weight of mice in anti-GITR m Ab group decrease was statistically significant(P<0.01,P<0.05).2.The gingival index of mice in the periodontitis group,the Ig G group and the anti-GITR m Ab group increased compared with the normal control group,with a significant difference(P <0.01).The anti-GITR m Ab group had a higher gingival index than the periodontitis group and the Ig G group,and the difference was statistically significant(P <0.05,P <0.01).Compared with the mice in the normal control group,the periodontitis group,the Ig G group and the anti-GITR m Ab group had significantly higher teeth mobility,and the differences were significant(P <0.01).The degree of tooth mobilityl in the anti-GITR m Ab group was greater than that in the periodontitis group and the Ig G group,the difference was statistically significant(P <0.01,P <0.01).3.The measurement of the distance from the enamel cementum junction to the alveolar crest showed that:compared with the normal control group,the distance between the enamel cementum junction to the alveolar crest of the mice in the periodontitis group,the Ig G group and the anti-GITR m Ab group increased(P <0.01).The distance from the enamel cementum junction to the alveolar crest of the anti-GITR m Ab group mice increased than the periodontitis group and the Ig G group(P <0.05,P <0.01).4.The results of hematoxylin-eosin staining showed that in the normal control group,the intraepithelial epithelium was intact,the combined epithelial position was normal,no inflammatory cell infiltration was found in the gingival epithelium,the periodontal ligament fibers were arranged neatly,and the alveolar bone edge was clear and intact.cell.The periodontitis group and the Ig G group: epithelial erosion or ulceration occurred in the sulcus,part of the epithelium infiltrated into connective tissue with inflammatory cell infiltration,and some inflammatory cells and exudates were removed into the periodontal pocket.Combined with the epithelium to proliferate and extend to the root,a periodontal pocket is formed,with inflammatory cells infiltration around it.The intraepithelial epithelium and the collagen fibers below the combined epithelium edema and degeneration,and some have been replaced by inflammatory cells.The alveolar crest and the inherent alveolar bone are absorbed.The anti-GITR m Ab group: epithelial erosion in the groove,most of the epithelium proliferated into connective tissue,a large number of inflammatory cells infiltrated,most of the inflammatory cells and exudates moved to the periodontal pocket,combined with the epithelium roots,forms a deep periodontal pocket.Combined with the loss of subepithelial collagen fibers,most have been replaced by inflammatory cells.Alveolar bone has obvious bone resorption pits,and a large number of osteoclasts can be seen around it.The inherent alveolar bone is seriously absorbed and damaged.5.The results of real-time quantitative RT-PCR showed that compared with the normal control group,the expression levels of Treg cell-associated factors IL-10,TGF-β1 and Foxp3 m RNA in the gum tissue of mice in the periodontitis group,Ig G group and anti-GITR m Ab group increased(P <0.05).The expression level of i NOS m RNA of M1-type macrophage-associated factor increased(P<0.01).Compared with the periodontitis group and Ig G group,the expression levels of Treg cell-associated factors IL-10,TGF-β1 and Foxp3 m RNA in the gingival tissue of the anti-GITR m Ab group were reduced(P <0.05),and M1 macrophages were associated with the expression level of factor i NOS m RNA increased(P <0.05).Conclusions1.In experimental periodontitis,the immune response mediated by M1 macrophages is enhanced,and M1 macrophages are involved in periodontal tissue inflammation and tissue damage.2.In experimental periodontitis,Treg cells can down-regulate the M1-type macrophage-mediated immune response and reduce the inflammation and tissue damage of periodontitis. |
