The Expression Of TRAF6 In Human Gingival Fibroblasts Inducted By IL-1β And LPS

Objective:①The stable Human gingival fibroblasts model will be constructed in vitro to provide the experiment conditions on the effect of cytokine network to HGFs.①The expression of tumor necrosis factor receptor-associated factor-6(TRAF6) will be observed in HGFs after the intervention of IL-1βand LPS to provide theoretical evidence to enrich the investigation of the effect of cytokine network to gingival tissue.Methods:①The primary cells were isolated from human gingiva and cultured by tissue explant culture technique, immunohistochemistry method was used to assess the cell with vimentin and ceratin, the proliferation discipline was observed via the growth curve, in order to know HGFs' biology features and construct the stable Human gingival fibroblasts model in vitro.②The cells from the third to eighth passage were cultured successfully to make cell seeded and stimulated with IL-1βof Ong/ml,0.1ng/ml,0.5ng/ml,1ng/ml,5ng/ml,10ng/ml or LPS of 0μg/ml,0.1μg/ ml,1μg/ml,10μg/ml,50μg/ml,100μg/ml on cells respectively. The morphology changes of HGFs were observed after 24 hours and the expression of TRAF6 in every experimental group was detected with immunohistochemical methods.The test results were collected and analyzed with computer image analysis system.The experiment data of the expression features of TRAF6 after intervention on HGFs with IL-1βand LPS was analyzed with single-factor variance analysis.Results:①The HGFs were cultured with tissue explant culture technique and the stable Human gingival fibroblasts model was constructed in vitro successfully. The cells showed long spindle and star appearance under invert microscope, the cells had a positive reaction to antibodies against vimentin and a negative reaction to antibodies against ceratin with immunocytochemical analysis, the cells showed the typical derived from mesoderm fibroblast-like cell characteristics; After subculture, through the observation of cell growth curve, the vigor of the cells from third to eighth passage were well, the speed of proliferation was high, shape of cells were in rule, and the cells after eighth appeared aging and apoptosis signs, the speed of proliferation slowed significantly.②The immunohistochemistry results of the expression of TRAF6 in HGFs with the intervention of IL-1βand LPS in the protein level showed that:TRAF6 in the control group shows a negative reaction, but shows a positive reaction in the cytoplasm of the cell after the intervention of IL-1βand LPS on HGFs.The result of the expression status of TRAF6 in HGFs with the intervention of IL-1βrevealed that:in the experimental range of concentration the expression of TRAF6 showed the increasing trend with the intervention of IL-1βconcentration increased. The result of the expression status of TRAF6 in HGFs with the intervention of LPS revealed that:the expression of TRAF6 showed the increasing trend with the intervention of LPS in 0.1μg/ml,1μg/ml,10μg/ml experimental range of concentration but showed the gradually decreasing trend with the intervention of LPS in 10μg/ml～100μg/ml experimental range of concentration; While the results of cellular morphology observation with the intervention of IL-1βand LPS on HGFs showed that:with the intervention of LPS in a concentration increased on HGFs the cell morphology changed gradually including the proportion of stellate cells increased and the cells gradually showed aging apopotis phenomenen. But with the intervention of IL-1βconcentration increasing, the chang of the cell morphology was not obvious.Conclusions:①The stable Human gingival fibroblasts model was constructed in vitro successfuelly with tissue explants culture technique, tissue explants culture technique is the most common primary culture method, and its steps simple, high successl rate and can reflect the biology characteristics of the original organization, the cells from third to eighth were suitable for the experiment study, the cells after the eighth were not suitable for experiment study with showing aging apopotis phenomenon.The study can provide the experiment basis on the effect of cytokine network to HGFs.②The result of expression of TRAF6 in HGFs with the intervention of IL-1βand LPS put forward that TRAF6 involvs in the occurrence and development of gingival desease, and plays an important role in regulation in the process of gingival tissue inflammation.