Effects Of Phenytoin On The Proliferation And Expression Of Tumor Necrosis Factor-α In Human Periodontal Ligament Fibroblasts Stimulated By Lipopolysaccharide

BackgroundPeriodontal disease seriously threats to the human health, the incidence of periodontal disease is also a trend of increasing year by year in our country.Periodontal disease is a chronic inflammatory disease, LPS is one of the causes of periodontal disease.PHT is one of the traditional antiepileptic drugs, for its adverse reactions-GO, and therefore there have been conducting of its experiment and clinical research on wound healing.Fight infection and wound healing complements each other, both are the key problems to solve wound treatment. Studies have shown that PHT can promote h PDLFs proliferation in vitro. LPS is a major pathogenic substances of periodontal disease, inhibiting the h PDLFs proliferation, inducting of the h PDLFs to secrete IL-6,TNF-α inflammatory factor,etc.Yet research about effects of PHT on the proliferation and expression of TNF-α in h PDLFs intervened by LPS is rare, so this experiment uses the different concentrations of PHT and LPS and h PDLFs to co-culture, observating of PHT on h PDLFs biology effect by LPS stimulation in vitro, discussing its role in controlling the periodontal inflammation, providing a certain basis for treatment of periodontal disease.Objective1.To study the culture method of human periodontal ligament fibroblasts in vitro. 2.To observe the effect of Phenytoin on the proliferation in human periodontal ligament fibroblasts intervened by LPS. 3.To observe the effect of Phenytoin on the expression of tumor necrosis factor-α in human periodontal ligament fibroblasts intervened by LPS.Methods1.modified enzymatic digestion tissue piece method was used to culture the human periodontal ligament fibroblasts. 2.MTT assay was used to observe the effect of Phenytoin on the proliferation in human periodontal ligament fibroblasts treated with LPS. 3. Immunocytochemistry method was used to observe the effect of Phenytoin on the expression of tumor necrosis factor-α in human periodontal ligament fibroblasts treated with LPS.ResultsBy using modified enzymatic digestion tissue piece method can cultivate the human periodontal ligament fibroblasts, the morphological identification of cells was proved to be long fusiform or spindle shape, a typical ibroblast-like cells;Identified by immunocytochemistry showed vimentin antibody staining positive,cytoplasm brown staining; keratin antibody staining negative,cytoplasm not staining.h PDLFs belongs to mesenchymal origin.1. The results of MTT method to measure cell proliferation experiment showed that different concentrations(0.01 mg/L-100 mg/L) of LPS can obviously inhibit the proliferation activity of h PDLFs;Suitable concentration(5 mg/L-100 mg/L)of phenytoin can promote h PDLFs proliferation activity, but too high concentration(500 mg/L) of phenytoin can inhibit the activity of h PDLFs proliferation.100 mg/L of phenytoin have an antagonistic effect on LPS,inhibition on the proliferation activity of h PDLFs. 2. Immunocytochemistry method results showed that the LPS can obviously increase the expression of TNF-α in h PDLFs and 100 mg/L of phenytoin intervention can significantly weaken the expression of TNF-α in h PDLFs.Conclusions1. Phenytoin(5mg/L-100mg/L)stimulated the proliferation of h PDLFs. 2. 100 mg/L phenytoin not only can modify the inhibition of LPS on the proliferation of h PDLFs,but can inhibit the expression of TNF-α in h PDLFs. 3. Phenytoin not only can control inflammation,but promote h PDLFs proliferation, the combination may be used for the treatment of periodontitis.