Study On The Apoptosis And Inflammatory Cytokine Induced By LTA And LPS On Human Periodontal Ligament Cells

Objective To investigate the effects of LTA and/or LPS on human periodontal ligament cells'apoptosis and inflammatory cytokines production.Methods Human periodontal ligament cells (hPDLC) that are prepared from the middle 1/3 periapical tissues were maintained in high glucose DMEM medium with or without LTA and/or LPS (1) Ultra-structure and apoptotic body were observed by transmission electron microscope; growth and proliferation were examined by MTT colorimetric assay; the expression of TNF-a was examined by enzymelinked immunosorbent assay (ELISA); quantitative analysis of apoptotic cells were evaluated by flow cytometry. (2) Real-time PCR was used to detect Fas, Bax, Caspase-3 and Cytochrome C mRNA expression, and Western Blot was utilized to determine the corresponding proteins in human periodontal ligament cells. (3) Inflammatory cytokines were measured by Antibody chip technology.Results (1) Condensesd and vacuolited cytoplasm and apoptotic body were observed in each group throμgh transmission electron microscope. Both LTA and LPS could stimulate apoptosis of hPDLC. TNF-a secretion can be detected 12h after stimulation with LTA and/or LPS and increasingly escalate within 48h. There are no statistic difference between 100μg/ml LTA group and 1μg/ml LPS group at the same time point. Apoptosis rate of early and late stage in each groups were obviously enhanced compared with control group throμgh flow cytometry technology. (2) Cytochrome C, Fas, and Caspase-3 mRNA were up-regulated throμgh real-time PCR after incubated with LTA and/or LPS, similarly, the production of corresponding proteins were also increased. Expression of Bax mRNA decreased. (3) Various cytokines were released by hPDLC after LTA and/or LPS stimulation, especially IL-1, which was closely related to the apoptosis process. LTA, LPS have synergistic effect, their combination can greatly enhance the expression of apoptosis-related cytokines such as TNF-a, p and IL-1.Conclusions (1) Both LTA and LPS can induce apoptosis of human periodontal ligament cells. (2) LTA and LPS can enhance the expression of TNF-a. (3) The production of Fas, Cytochrome C, Caspase-3 mRNA and protein were up-regulated when induced by LTA and/or LPS in hPDLC. Expression of Bax mRNA decreased. (4) The types of up-regulated cytokines were similar when induced by LTA and LPS respectively, but the effect of the former was relatively inferior. (5) LTA and LPS have synergistic effect; their combination can greatly enhance the expression of apoptosis-related cytokines.