| Objective:Periodontitis is an inflammatory disease of the supporting tissues of the teeth caused by specific microorganisms in plaque,resulting in progressive destruction of the periodontal ligament and alveolar bone with periodontal pocket formation, attachment loss,progressive alveolar bone absorption and teeth loss.The mechanism of periodontitis is complex and it is necessary to farther researches.High mobility group protein B1(HMGB1) widely exists in eukaryotic nucleus which is one of the the most important non-group protein in the regulation of gene expression.It has been found that HMGB1 has a variety of biological functions in and out nuclears in recent studies,especially as a wide range of cytokines involved in inflammatory response.It aroused world wide attention in many research fields. HMGB1 is released from activated monocytes to participate in lethal effects,and can activate the release of downstream cytokines.Studies of delayed kinetics of HMGB1 demonstrated that it was a potential therapy as the factor by the targeting of cytokines in the future.So far,little is known about HMGB1 on the role in periodontitis.This study is in order to fill this gap.Therefore,the experiments are designed to observe the effect of HMGB1 on human periodontal ligament fibroblasts in vitro and to preliminary study HMGB1 in the pathogenesis of periodontal disease.Methods:Periodontal ligament fibroblasts were collected from the orthodontic extraction of healthy premolars' periodontal ligament tissue.And tissue was cultured with DMEM medium.After reached confluence,cells were passaged and the generation among 3～6 cells were used in the experiment.1.The 1×10~4 cells /ml cell suspension were inoculated into the pre-placed piece of cells mounting in 6-well plates.With vimentin and keratin antibody as primary antibodies,ABC immunohistochemical staining was used to identify the source of human periodontal ligament fibroblasts cells.2.The 2×10~4 cells /ml cell suspension were put in 96-well plates and inoculated 24h with the high mobility group protein B1(0,10,30,100,500,1000ng/ml) individully.After cells challenaged with 8h,12h,24h,48h,the mothed of MTT was adopted to assay cells' proliferation situation.3.The 1×10~6 cells/ml cell suspension were put in 6-well plates inoculated 24h with HMGB1(0,10,30,100,500,1000ng/ml),IL-6,RANKL,OPG expression of the mRNA in cells were detected by RT-PCR.4.The periodontal ligament fibroblasts were challenaged with HMGB1 after 24h(0,10,30,100,500,1000ng/ml)in culture bottles,RANKL,OPG protein expression in cell were detected by Western Blot.Results:1.After the identification of immunohistochemistry,the cultured cells showed anti-vimentin protein positive,and anti-keratin negative,which proved that the cultured cells were derived from mesodermal fibroblasts.2.MTT results showed that challenaged with 30ng/ml concentration HMGB1 at 24h,periodontal ligament cells proliferated significantly(P＜0.05).But 500ng/ml concentration at 24h the proliferation of periodontal ligament cells were inhibited (P＜0.05).1000ng/ml concentration at 24h,48h,the proliferation of periodontal ligament cells were inhibited(P＜0.05).3.RT-PCR results showed that HMGB1 in the concentration of 10ng/ml, RANKL/OPG ratio increased significantly(P＜0.05),100ng/ml concentration of IL-6mRNA expression was significantly higher(P＜0.05).4.Western Blot results showed that,in 10ng/ml concentration group, RANKL/OPG ratio were also higher than that in the control group. Conclusion:Low concentration(30ng/ml) of HMGB1 made periodontal ligament cells proliferation,but reaching a certain high concentration(500ng/ml,1000ng/ml),it inhibited human periodontal ligament cells' proliferation.10ng/ml HMGB1 could promote human periodontal ligament cells to the direction of differentiation of osteoclasts,and 100ng/ml HMGB1 could induce periodontal ligament cells to increase the expression of IL-6mRNA.These showed that HMGB1 might be the incidence of periodontitis and inflammation and it could play an important role in promoting progress.
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