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Purpose by the in vitro culture of human periodontal ligament cells (human periodontal ligament cells, HPDLCs), and by observing lipopolysaccharide (lipopolysaccharide, LPS) HPDLCs proliferation and secretion of tumor necrosis factor-α (tumor necrosis factor-α, TNF-α) interferon-gamma (interferon-γ, IFN-γ), interleukin-β (interleukin-1β, IL-1β) and leukocyte interleukin -8 (interleukin-8, IL-8) the impact of observed LPS effect HPDLCs After the nuclear transcription factor-kB (nuelearfactor kappa B, NF-kB) expression, and to explore the role of LPS in the pathogenesis of periodontitis, provide a theoretical basis for new ways to find prevention and drug treatment of periodontitis. Method to collect 12-28 years old molars before orthodontic extraction of the health of the 60 cases, the use of tissue blocks directly adherent culture method vitro culture HPDLCs, and pedestrians and periodontal ligament cell source identification. Will HPDLCs divided into two groups, i.e., the experimental group and the control group: the control group were placed in containing different concentrations of LPS (experimental group: 0.01μg/ml, 0.1μg/ml, 1μg/ml, 10μg/ml, and 100μg/ml; 0μg / ml) in the culture medium after 24h, the MTT assay HPDLCs proliferation. Selection 10μg/ml LPS role HPDLCs 24h after LPS effect HPDLCs before and after alkaline phosphatase (alkaline phosphatase, ALP) activity were detected. Selection 10μg/ml the LPS stimulation HPDLCs, and enzyme-linked immunosorbent assay (enzymelinked immunosorbent assay, ELISA) were measured before LPS stimulation and stimulus 3h, 6h, 24h, 48h after cell supernatant of TNF-α, IFN-γ ), IL-1β, IL-8 expression. Selection 10μg/ml LPS stimulation HPDLCs 24h, immunofluorescence assay before and after LPS stimulation HPDLCs in the expression of NF-kB. The diversity of this number were compared using one-way ANOVA, two sample t-test was used to compare. Test α = 0.05, P lt; 0.05 result is a statistically significant difference. Vitro culture HPDLCs60 cases, success of the 15 cases, 25% success rate. Cultivated anti-cytokeratin-negative, anti-osteopontin (osteopontin, OPN) positive staining cells, to prove cell non-epithelial origin, and has the characteristics of osteogenesis by immunohistochemical staining. 2 below 1μg/ml concentration LPS promote HPDLCs proliferation, which promote the most obvious effect of the concentration of 0.01μg/ml (P LT; 0.05), decreased gradually increasing concentration role; LPS - concentration of 10μg / ml and 100μg/ml HPDLCs proliferation was not affected (P gt; 0.05). The ALP activity in 3.LPS after 24h HPDLCs decreased significantly (P lt; 0.01). 4.LPS stimulus HPDLCs 3h can be detected TNF-α, IFN-γ and IL-1β secretion volume was significantly higher (P lt; 0.05), and with the role of time gradually increased, peaked to 24h; subsequent three cytokines secretion volume decreased to 48h of IFN-γ and IL-1β still maintain a high level (P lt; 0.05), and TNF-α secretion recovered to the pre-stimulus level (P gt; 0.05 ). LPS stimulation HPDLCs3h, and 6h detect IL-8 secretion was significantly inhibited (P lt; 0.05), and subsequently, the time-dependent increase until 24h after IL-8 secretion was higher than the pre-stimulus level 48h peaked (P lt; 0.05). Immunofluorescence detection NF-kB 5.LPS role mainly expressed in the cytoplasm, with strong fluorescence in the cytoplasm of the weak nuclear fluorescence; LPS, NF-kB nuclear translocation in the nucleus the fluorescence significantly strengthened, weakened cytoplasm fluorescence. Conclusion 1. Organizations block culture method simple and high success rate HPDLCs 4-8 cells with high proliferative activity, applicable in vitro studies. Directly 2.LPS affect HPDLCs proliferation, inhibit their osteogenic characteristics, and can stimulate HPDLCs caused by the secretion of inflammatory cytokines, LPS is an important virulence factors of periodontal inflammation and alveolar bone resorption. 3.LPS can stimulate HPDLCs secrete a variety of inflammatory cytokines, and has a certain relationship between the aging, the temporal relation of different inflammatory cytokines. 4.LPS the stimulation can HPDLCs occurrence obvious nuclear translocation of NF-kB, prove that NF-kB in LPS-induced periodontal inflammation reaction.
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