Effect Of Carboxymethyl Chitosan On The Expression Of PGE₂and IL-6in Human Periodontal Ligament Fibroblasts Induced By LPS

Objective:The purpose of this study is to cultured human periodontal ligament fibroblasts basis in vitro, utilized the immunohistochemical method to discover the influence of Carboxymethyl Chitosan on LPS on human periodontal ligament fibroblast cells expressed PGE2and IL-6. And to investigate the carboxymethyl chitosan in the treatment of periodontitis mechanism and feasibility, provide a new way for the treatment of periodontal disease and a laboratory evidence for further searching for periodontal disease drug.Methods:①Revived the human periodontal ligament fibroblast, the cell morphology was observed under microscope, immunohistochemical method was used to detect the expression of cytokeratin and filamin, determined their origin, and established a stable human periodontal ligament fibroblast model.②With selected the4-5cultured cells,after climbing films, different concentrations of LPS were used on cell, with PBS as the negative control group, to observe the change of24h after each cell morphology.The expression changes were detected by immunohistochemical staining of PGE2and IL-6.The results were based on the analysis system result of medical image, and screened the optimum concentration of LPS intervention.③A concentration on human periodontal ligament fibroblast model of optimal LPS intervention,used different concentrations of Carboxymethyl Chitosan(Omg/L,0.1mg/L,1mg/L,10mg/L,50mg/L,100mg/L) and the best intervention concentration of LPS on human periodontal ligament fibroblasts,the changes were observed after24h.Then detected the expression changes of PGE2and IL-6using immunohistochemistry, image analysis and data collection on the results used of medical image analysis system.By using analysis of variance to observe the statistical data of PGE2and IL-6expression characteristics. Result:①The frozen resuscitated cells’sculture fluid is clear without impurities and no abnormal substances under microscope.The cells are long fusiform and star shaped. Cell body is round, cytoplasm is homogeneous, the nucleus round or oval. Anti vimentin polyclonal antibody (VIM) positive expression, anti cytokeratin (CK) staining negative expression in cells, which confirm that the cells derived from mesenchymal tissue, while the non epithelial tissue.②After LPS stimulation HPDLFs in different concentration, with the increase in concentrations of LPS intervention, the cytoplasmic staining first increased and then reduced, and when LPS concentrations is10mg/L, cytoplasmic staining of expression of PGE2and IL-6are strongest positive, statistically significant differences (P<0.05).③Carboxymethyl Chitosan with different concentrations were reduced in the best concentration (10mg/L) LPS intervention in human periodontal ligament fibroblasts expressing PGE2and IL-6, and with increasing concentration of carboxymethyl Chitosan,the expression of PGE2and IL-6show a gradual downward trend, results showed statistically significant differences(P<0.05).Conclusion:①Recovery of cultured human periodontal ligament fibroblasts in vitro model is simple operation, low pollution rate, clear source, notable biological characteristics, which can be used for experimental study.②LPS intervention in human periodontal ligament fibroblasts to produce PGE2and IL-6clearly,shows PGE2and IL-6involved in the occurrence and development of periodontitis, and is the important cytokines in the process of developing periodontitis, thus these two factors may play a role in investigating the regulation of Carboxymethyl Chitosan on periodontitis.③PGE2and IL-6play an important expression of inhibitory effect of Carboxymethyl Chitosan on LPS on human periodontal ligament fibroblasts inflammatory damage process.Thus Carboxymethyl Chitosan in periodontal inflammation plays an important role in the development process, which will provide new experiments for clinical drug treatment for periodontal disease information.