| Objective To explore the effects of lipopolysaccharide(LPS) on periodontitis by using the culture technique of human periodontal ligament cells(HPDLCs) in vitro and observing the influence of LPS on the proliferation and secreting tumor necrosis factor-α(TNF-α),interferon-γ,(IFN-γ,),interleukin - 1β(IL- 1β) and interleukin -8 (IL-8) stimulated by LPS.Moreover,the change of the expression of nuclear factor-kappa B(NF-kB) was investigated.Accordingly the theoretics basis was provided to find a new method to prevent and treat periodontitis.Methods 60 healthy premolars extracted for orthodontic reasons from 12 to 28 years old were selected and periodontal tissues were collected and cultured in vitro to obtain HPDLCs.After the 4th passage HPDLCs being cultured for 24h in DMEM medium containing LPS with the concentration as follow:0μg/ml,0.01μg/ml, 0.1μg/ml,1μg/ml,10μg/ml and 100μg/ml respectively,the proliferation of HPDLCs were measured by MTT assay.Then 10μg/ml LPS was choosed to stimulate HPDLCs for 24h and the alkaline phosphatase(ALP) activity of HPDLCs was measured.The TNF-α,IFN-γ,IL-1βand IL-8 concentrations in cell supernatants were detected by sandwich ELISA at the 3h,6h,24h and 48h after 10μg/ml LPS stimulating, respectively.The expression of NF-kB was determinded with immunofluorescence before and after being cultured with 10μg/ml LPS for 24h.Comparison of the mean varied using one-way ANOVA,the two samples were compared using t test.The test standard wasα=0.05,and the results in P<0.05 were that differences with statistical significance.Results1.HPDLCs can be obtained in vitro successfully by tissue culture.2.The influence of LPS on HPDLCs is related to its concentration.LPS with concentration below 1μg/ml promoted the proliferation of HPDLCs,the most obvious concentration was 0.01μg/ml(P<0.05),and the effect was decreased with concentration of LPS increasing.It was almost no effect of LPS with concentration 10μg/ml and 100μg/ml on HPDLCs(P>0.05).3.The alkaline phosphatase activity was decreased significantly with LPS for 24h (P<0.01).4.After being stimulated by effective concentration of LPS with 10μg/ml,the secretion of TNF-α,IFN-γ,and IL-1βwere increased significantly for only 3h (P<0.05),moreover the effects were increased gradually with the time,the peaks were at 24h.Then the secretions of all three cytokines decreased.The secretion of IFN-γ,and IL-1βwere maintained at a high level still at 48h(P<0.05),however the secretion of TNF-αwas come back to the level before stimulation(P>0.05).The secretion of IL-8 differed to the other three cytokines.It was inhibited significantly at 3h and 6h(P<0.05),and then the secretion of IL-8 started to increased with time-dependence,it was detected higherly than the level before stimulation at 24h, and the peak appeared at 48h(P<0.05).5.The role of NF-kB was mainly expressed in cytoplasm before being stimulated by LPS,the cytoplasm showed strong fluorescence,a weak nuclear fluorescence; after LPS stimulation the expression of NF-kB occurred nuclear translocation obvious, obvious fluorescence in the nucleus strengthened,weakened fluorescent cytoplasm.Conclusion1.Tissue culture of human periodontal ligament cells cultures easily and the success rate is high relatively,the first 4th-8th passage cells with high proliferative activity are fit to experimental research related in vitro.2.E.coli LPS can be successfully induced inflammation model of human periodontal ligament cells.LPS may impact HPDLCs proliferation directly,and stimulate HPDLCs to secrete inflammatory cytokines,inhibit its osteogenic characteristics.So that LPS is an important virulence factor to cause the absorption of alveolar bone and periodontal disease.3.LPS can stimulate HPDLCs to secrete various inflammatory cytokines,and the effect is related to time.Different inflammatory factor have different relationship with time.4.LPS can cause HPDLCs to occur NF-kB nuclear translocation significantly that NF-kB is involved in the LPS-induced response of periodontitis.
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