Expression Of IL-1β,IL-6 And TNF-α Induced By Actinomyces Viscosus Fimbriae In Fibroblasts

Actinomyces viscosus appears to be a key organism associated with periodontal destruction in patients with periodontitis. Many studies indicated that Actinomyces viscosus fimbriae may be an important cell structure involved in triggering the pathogenesis brought about by organism, because the fimbriae promote the attachment of the organism to periodontal tissues. However, Immunological characterizations of Actinomyces viscosus fimbriae have not been examine detail. Therefore, the research about the effection of Actinomyces viscosus fimbriae on some cytokines expression is important to demonstrate the pathogenic mechanisms of the organism in periodontal disease.Objective:to obtain Actinomyces viscosus fimbriae and examine whether Actinomyces viscosus fimbriae, an important structure involved in attachment of the bacteria to periodontal disease , active fibroblasts and subsequently induce production of TNF-α,IL-1β,IL-6.Methods:First, Actinomyces viscosus fimbriae were obtained by homogenization and centrifugation. Chemical analysis, amino acid analysis and electron microscopy analysis of the fimbriae were done to detect the purity of fimbriae.Then, Fibroblasts were treated with the fimbriae. After 48 hours, the culture mediums were collected. TNF-α,IL-1β,IL-6 in the culture mediums were detected by Western-blotting.Result:1. Electron Microscopy result :(1) Numerous fimbriae were detected on the surfaces of Actinomyces viscosus that were cultured anaerobically for 48 hours.(2) Fimbriae were removed from the surfaces of Actinomyces viscosus through homogenization and centrifugation.2. Chemical analysis and amino acid analysis of the fimbriae: The fimbriae were mainly composed of protein and small amount of carbohydrate.The fimbeiae contained large amounts of glutamic acid,glycine,arginine and high percentage of nonpolar amino acid .3. Western-blotting result:(1)The proteins were transferred from SDS-PA gel to the nitrocellulose membrane and were detected with anti-TNF-αmonoclonal antibody from goat followed by antigoat–IgG-conjugated with alkaline phosphatase. The result showed that TNF-аexist in culture medium treated with fimbriae, but TNF-аare not found in culture medium without fimbriae.(2) The proteins were transferred from SDS-PA gel to the nitrocellulose membrane and were detected with anti-IL-1βmonoclonal antibody from rabbit followed by antirabbit–IgG-conjugated with alkaline phosphatase. The result showed that IL-1βexist in culture medium treated with fimbriae, but IL-1βare not found in culture medium without fimbriae.(3) The proteins were transferred from SDS-PA gel to the nitrocellulose membrane and were detected with anti-IL-6 monoclonal antibody from goat followed by antigoat–IgG-conjugated with alkaline phosphatase. The result showed that IL-6 exist in culture medium treated with fimbriae, but IL-6 are not found in culture medium without fimbriae.Conclusion:1. Maximum removal of the Actinomyces viscosus fimbriae occurred though homogenization and centrifugation.2. Observations suggest that Actinomyces viscosus fimbriae could trigger TNF-α,IL-1β,IL-6 production by fibroblasts...