The Expression Of TRAF6 In Human Periodontal Ligament Fibroblasts By IL-1β Intervention

[Objective]: To isolate and culture the human periodontal ligament fibroblasts (HPLFs), construct the cell model in vitro; and research the expression of tumor necrosis factor receptor - associated factor-6(TRAF6) in HPLFs by the interference of IL-1β, and provide new document to the research of the effect of cytokine network to cells.[Method]: The primary cells were isolated from human periodontal ligament and cultured by tissue explant culture technique, then passaged when the cells grew in the bottom of culture flasks completely. Morphological analysis and immunohistochemistry analysis were used to characterize the cell lineage. Choose the fifth to eighth passage and stable growth of cells. And use IL-1βof 0ng/ml,0.1ng/ml,0.5ng/ml,1ng/ml,5ng/ml,10ng/ml on cells intervention respectively. Using reverse transcription-polymerase chain reaction(RT-PCR) and immunohistochemical methods to detect the expression of TRAF6 , and collect the test results ,include image and data by computer image analysis system. The experiment data using single-factor variance Analysis for statistical analysis, to observe the expression and location of TRAF6 in different concentrations of IL-1βintervention.[Result]: (1) The HPLFs were cultured and passaged successfully. After the fifth passage, cells grew prosperity and show long spindle appearance, are a typical fibroblast-like morphology. Through immunocytochemical analysis, the cells had a positive reaction to antibodies against vimentin, and a negative reaction to antibodies against ceratin. It certified the cells were fibroblasts from mesoblast, and in line with human periodontal ligament fibroblasts in vitro biological properties. (2) The results of RT-PCR and immunohistochemistry showed that: in the gene and protein level, TRAF6, in the normal HPLFs shows a negative reaction, but shows a positive reaction in the cytoplasm of the cell by IL-1βintervention, and the intensity of its expression of IL-1βintervention has strengthened by the trend of concentration.[Conclusion]: (1) Culture cells by the method of tissue explants, steps simple and easy to grasp, the cells' morphological and immunocytochemical characteristics cultured by tissue explant culture technique were consistent with those of typical HPLFs. The growth condition of the cells was satisfied. The cells can be a model of HPLFs in vitro for future research about regulation affect that cytokines to the cells function. (2) In gene and protein level, the experiment has proved TRAF6 involved in the signal transduction process in the HPLFs mediated with EL-1β, as a network of cytokines involved in HPLFs function regulation and control, provides a new idea to periodontal disease diagnosis and treatment.