| The human periodontal ligament fibroblasts (HPDLFs) is a kind of highly specialized connective cells, which excrete extracellular matrix to connect the cementum with the alveolar bone. HPDLFs play a major role in alveolar bone metalolism in periodontal health or disease, for their ability to secrete factors that regulate the homeostasis of connective and osseous tissue, including the major osteoclast regulators RANKL and OPG. HPDLFs express receptor activator of RANKL, which blinds to the receptor activator of NF-kappa B(RANK) and is required for osteoclast maturation. Farther more, HPDLFs also secrete OPG, which is a soluble member of the tumor necrosis factor (TNF) receptor ruperfamily and join in the regulation of bone density. So it has been believed that the balance of these two factors regulated by HPDLFs is critical for alveolar bone destruction during periodontitis. Many cytokines have been reported to play an important role in the pathogenesis of periodontitis, such as tumor necrosis factor-α(TNF-α)and prostaglandin E2 (PGE2 ).These cytokines cause inflammation and destruction of tissue and resorption of alveolar bone. Although the cytokines cause the periodontitis via stimulating HPDLFs, but the cellular and molecular mechanisms of TNF-αand PGE2 in HPDLFs haven't been understood completely.Therefore, in the present study, we examined the effects of TNF-αand PGE2 on the expression of RANKL/OPG mRNA in HPDLFs by semi-quanitative RT-PCR and fluorescent quantitative RT-PCR.The result show that TNF-αand PGE2 stimulate HPDLFs'expression level of RANKL versus OPG, and suggest that TNF-αand PGE2 may join in the alveolar bone destruction by regulating HPDLFs.Objective:To explore the effect of cytokines on HPDLFs, this study detected the gene expression of RANKL and OPG in HPDLFs via the induction of TNF-αand PGE2. Methods:1.Cell culture and identification: HPDLFs were isolated and cultivated from periodontal ligament of third molars and premolar teeth by tissue explants method. Their morphology and growth condition were observed by light microscope.Immunocytochemical stain were used to characterize the cell lineage.2. Effect of TNF-αon the expression of RNAKL and OPG mRNA in cell: The fifth generation HPDLFs were used to experiment, and were inducted in media containing 1100 ng/ml of TNF-α.After 24-hour incubation, semi-quanitative RT-PCR was performed to detect the expression of OPG and RANKL mRNA usingβ-actin mRNA as the internal control.3. Effect of PGE2 on the expression of RNAKL and OPG mRNA in cell: HPDLFs were cultured in media containing 10-910-6 mol/L PGE2. After incubation for 248h, semi-quantitative RT-PCR was performed to detect the expression of OPG and RANKL mRNA usingβ-actin mRNA level as the internal control.4. Effect of TNF-αand PGE2 combination on the expression of RNAKL and OPG mRNA in cell: HPDLFs were cultured in media containing 1100 ng/ml of TNF-αand 10-810-6 mol/L of PGE2.After 24-hour incubation,fluorescent quantitative RT-PCR (FQ-RT-PCR) was performed to detect the expression of OPG and RANKL mRNA usingβ-actin mRNA as a internal control.Results:1. HPDLFs were successfully isolated and cultured from the human periondontal legament,and were certified by immunocytochemical stain.Cultured cells were spindle-shaped,and had a positivere action to antibodies against vimentin,and a negative reaction to antibodies against keratin.2. TNF-αup-regulated the mRNA expression of RANKL, and increased the RANKL/OPG ratio in human periodontal ligament fibroblasts.3. 10-9-10-6 mol/L PGE2 up-regulated the mRNA expression of RANKL but inhibited the mRNA expression of OPG, and increased the RANKL/OPG ratio in human periodontal ligament fibroblasts .4. 10 ng/ml of TNF-αand 10-7 mol/L of PGE2 combination significantly up-regulated the mRNA expression of RANKL, and increased the RANKL/OPG ratio in human periodontal ligament fibroblasts.Conclusions1. TNF-αand PGE2 can respectively and cooperatively stimulate HPDLFs'expression level of RANKL versus OPG, and suggest that these inflammatory cytokines may join in the alveolar bone destruction by regulating HPDLFs'cellular and molecular mechanisms.2. 10 ng/ml TNF-αand 10-7 mol/L PGE2 were cooperative interactions on the gene expression of RANKL/OPG in human periodontal ligament fibroblasts.
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