Effects Of RhIL-1α And 1,25-(OH)2D3 On The Expression Of RANKL And OPG In Human Periodontal Ligament Fibroblasts Cells

Bone metabolism is a balance of bone formation and bone resorption. There are many factors that influence the balance of bone metabolism which including mechanical strain, the activities of cells and so on. In periodontal tissue, not only the osteoblast and osteoclast, but also the periodontal ligament cells take part in the remodelling of alveolar bone. Periodontal ligament cells (PDLCs) is a heterogenicitic cell mass, in which some cells can differentiate into osteoblasts and cementoblasts. Human periodontal ligament fibroblasts (HPDLFs) are the main cells of PDLCs which have features of osteoblasts. Furthermore, HPDLFs can express osteoprotegerin (OPG) and receptor activator nuclear factor kappa B ligand (RANKL) to regulate the activity of osteoclast. The immediate contact of osteoblast/matrix cells and osteoclast precursors (OCP) play an essential role in formation and differentiation of osteoclast (OC). Moreover, when osteoblast/matrix cells are exposed to a bone resorption stimulus, RANKL's binding with receptor activator of nuclear factor-κB receptor (RANK), which is expressed on the surfaces of OCP and OC, lead to a cascade reaction to promote the OC differentiation maturity and activation. Additionally, osteoblast/matrix cells secreted OPG and when these cells were cloned and studied, it was inhibit OC differentiation and its activities by competing with the binding of RANKL to RANK. So the ratio of RANKL to OPG is a lever of bone metabolism. HPDLFs through RANKL-RANK-OPG pathway, helps to regulate the remodelling of alveolar bone. Among the factors influence the activities and functions of HPDLFs. RhIL-1αand 1,25-(OH)₂D₃ are the important regulators which play an imortant role in alveolar bone remodelling. In the past study, rhIL-1αand 1,25-(OH)₂D₃ regulate the expression of RANKL and OPG in osteoblast and change the ratio of RANKL to OPG. The aim of this study was to observe the effects of rhIL-1αand 1,25-(OH)₂D₃ on the expression of RANKL and OPG in HPDLFs, to expand our knowledge in alveolar bone metabolism. In this study, HPDLFs were cultured in vitro and identified. Then, we planned to detect the expression of RANKL and OPG in normal HPDLFs and to investigate the influence of rhIL-1αand 1,25-(OH)₂D₃ on the expression of RANKL and OPG in HPDLFs.The study was divided into two parts as follows:一,Culturing HPDLFs in vitro and detecting the expression of RANKL and OPG in normal HPDLFsObjective(1) To establish study model: culturing HPDLFs in vitro and identifing the cells.(2)To detect the expression of RANKL and OPG from normal HPDLFs in transcriptional and protein level.(3) To discuss the regulated pathway of RANKL/OPG in alveolar bone remodeling.MethodsHPDLFs were cultured by using tissue explant culture technique and identified in vitro. Then, the expression of RANKL and OPG mRNA was detected by fluorescent quantitative RT-PCR (FQ-RT-PCR).The expression of RANKL and OPG protein was detected by immunocytochemistry and ELISA.Results(1) HPDLFs were cultured and identified in vitro.(2) HPDLFs express RANKL and OPG in transcriptional level. The positive expression of RANKL protein was detected on cellular membrane and endochylema of HPDLFs; The expression of OPG protein was also found to be expressed by HPDLFs.ConclusionHPDLFs can express OPG and RANKL in base condition. They are act via RANKL/OPG pathway to regulate the alveolar bone remodelling. Furthemore, since the OPG expression was stronger than RANKL, so the stability of periodontal ligament can be maintained.二,Effects of rhIL-1αand 1,25-(OH)₂D₃ on the expression of RANKL and OPG in HPDLFsObjective(1) To investigate the effects of rhIL-1αon the expression of RANKL and OPG in HPDLFs.(2) To evaluate the effects of rhIL-1αand 1,25-(OH)₂D₃ on the expression of RANKL and OPG in HPDLFs and to discuss the change of ratio of RANKL/OPG.MethodsTo investigate the influence of different concentrations of rhIL-1α(0,5,10,20ng/ml) on the expression of RANKL and OPG in HPDLFs by using FQ-RT-PCR HPDLFs were treated with 10ng/ml rhIL-1α, 1×10^-8mol/L1,25-(OH)₂D₃ or 10ng/ml rhIL-1αplus 1x10-8mol/L 1,25-(OH)₂D₃ for 48 hours. The expression of RANKL and OPG was measured by FQ-RT-PCR.Results(1) The expression of RANK and OPG was significantly influenced by the concentration of rhIL-1α. RhIL-1αwas found to be able to up regulate the expression of RANKL and OPG (F=48.870, P<0.05). However, the expression of RANKL was higher than OPG. So the ratio of RANKL/OPG were increased. In addition, the maximal effect on the ratio of RANKL/OPG was detected at a concentrations of 10ng/ml ( P<0.05).(2) RhIL-1αand 1,25(OH)₂D₃ influenced the expression of RANKL and OPG in HPDLFs. The expression of RANKL and OPG were increased with treated with rhIL-1α(P<0.05). Moreover, 1,25(OH)₂D₃ up regulate the expression of RANKL and decreased the expression of OPG, so the ratio of RANKL/OPG were increased too. (P<0.05). There was cumulative effect on regulate the expression of RANKL with the synergistic use of rhIL-1αand 1,25(OH)₂D₃ , but there was no cumulative effect on the ratio of RANKL/OPG (P>0.05). Conclusion(1) RhIL-1αhave close relation with alveolar bone remodelling. It not only up regulate the expression of RANKL, but also up regulate the expression of OPG. However, it increased the ratio of RANKL/OPG.(2) RhIL-1αand 1,25-(OH)₂D₃ act through RANKL-OPG pathway to regulate the alveolar bone remodelling. There was additive effect on the expression of RANKL with combination use of rhIL-1αand 1,25-(OH)₂D₃, but there was no additive effect or synergistic effect on the ratio of RANKL/OPG.