1.The Study Of DNA Methylation Profile Of Different Differentiation Stage Of MC3T3-E1 2.The Mechanism Study Of Osteoclastogenesis Inhibition By Taurine

Objective:Construction of the methylation profile of MC3T3-E1 cell line during it's proliferation, extracellular matrix maturation and mineralization stage. Analysis of the effect of mRNA expression caused by DNA methylation and demethylation.Methods:MC3T3-E1 cell line was cultured with medium contain ascorbic acid and (3-Glycerophosphate disodium. The mRNA expression of type I collagen (Collal), osteocalcin(Ocn), osteopontin(Opn), alkaline phosphatase (ALP) after cultured for 0 day,10day,24day were validated by real-time PCR. The morphology of cell was observed and the characteristic mineralized node staining was performed. gDNA of MC3T3-E1 cell line after cultured for 0 day, lOday,24day were extracted, and methylation microarray was performed to obtain the methylation profile of the three stages. Hyper-and hypo-methylated genes were chosen for stage-differential expression analysis. Differentially expressed genes were counted according to the chromosome distribution. By using DAVID online gene function and classification tool, the differentially expressed genes were analyzed for gene function classification and annotation. Validation of the profile result with the DNA methylation quantitative analysis of MassARRAY technology were carried out. The relationship between DNA methylation and gene expression is further confirmed with Real-time PCR for the differentially expressed genes. The DNA methyltransferase inhibitor RG108 was applied to intervene the MC3T3-E1 of proliferation stage, MTT and Real-time PCR of the differentially expressed genes were followed, and control group(with out RG108) were also carried out to validate its potential relationship.Result:1.The Real-time PCR result showed Colla1 expression of day 24 significantly increased compared to day 0 and day 10 after cultured with medium contain ascorbic acid,β-Glycerophosphate disodium of MC3T3-E1 cell line. The highest ALP expression was examined during day 10.The expression of Opn and Ocn is significantly increased in time series. The morphology of MC3T3-E1 in day 0 showed its spindle-shape whereas in day 10 it showed the "pavement stone" characteristics. After cultured for 22 days the cell grew to form multiple layers. Typical mineralized node could be seen with Von Kossa Staining after day 24. The differentiation stage of MC3T3-E1 could be classified as proliferation stage(day 0), extracellular matrix maturation stage (day 10) and mineralization stage(day 24) based on the expression of Colla1, ALP, Ocn and Opn.2. gDNA of the MC3T3-E1 of 3 stages were extracted to perform DNA methylation microarray profile. The hyper-methylated genes of proliferation stage, extracellular matrix maturation stage and mineralization stage counted 50,96 and 52, respectively. The hypo-methylated genes of the 3 stage counted 93,43, and 147, respectively. These differentially expressed genes were mainly involved in biological processes such as cell death, transcription regulation, metabolism, transport and signal transduction. Camkld, Bmprlb, Fbln7, Itga3, Pias2, Pcdhal2, Mdm4, Tmem54 mRNA expression were negatively correlated with methylation statue. Under the intervention of DNA methyltransferase inhibitor RG108, the hypermethylated genes expression were significantly upregulated.Conclusion:Cultured MC3T3-E1 cell line in day 0, day 10 and day 24 could represent the stage of proliferation, extracellular matrix maturation and mineralization stage, respectively. DNA methylation microarray captured the DNA methylation profile of the 3 stages. Analysis of the profiles showed the differentially expressed hyper-and hypo-methylated genes are involved in board range of biological process and function. During the differentiation process of MC3T3-E1 cell line, The methylation of promoter region caused down-regulation of the osteogenesis-related gene. RG108, the DNA methyltransferase inhibitor, could promote the proliferation of MC3T3-E1 cell line and upregulate the expression of DNA hypermethylated genes. Objective:Taurine is widely distributed in mammalian plasma and tissues, it is the most important free beta-amino acids in mammals. Our previous studies have found that Taurine could inhibit osteoclastogenesis, but the exact mechanism is still unknown. In this study, osteoblasts and bone marrow cells were co-cultured, in order to investigate whether Taurine inhibits osteoclastogenesis by affecting the expression of OPG and RANKL in osteoblasts.Methods:Skull of 24 hour newborn mouse and long bone of 6-8week aged mouse were isolated for osteoblast and bone marrow cell, respectively.The osteoblast and bone marrow cells were then co-cultured with different concentrations of Taurine for 6 days TRACP staining were performed for validation and counting of the osteoclasts. The expression of OPG and RANKL in osteoblast were then validated by real-time PCRResults:Taurine dose-relatively inhibited osteoclastogenesis in co-culture of osteoblasts and bone marrow cells, but Taurine showed no influential effect to the expression of OPG and RANKL in osteoblasts.Conclusion:Taurine inhibits osteoclastogenesis in a dose-dependent manner in co-culture of osteoblast and bone marrow cell. This inhibitory effect had no relationship with respect of OPG and RANKL in osteoblast.Objective:Our previous study found that RAW264.7 monocyte cell line expressed functional TAUT(taurine transporter), and Taurine could inhibit its differentiation. However, the relevance between the two is not clear. In this study, mature osteoclasts were obtained from M-CSF and RANKL induced bone marrow-derived macrophages, TAUT mRNA and Protein expression was measured by RT-PCR and immunocytochemistry. TAUT was blocked and then measured the Taurine effect on osteoclasts.Methods:Mature osteoclasts were obtained from M-CSF and RANKL induced bone marrow-derived macrophages. TAUT mRNA and Protein expression was measured by RT-PCR and Immunocytochemistry. RAW264.7 is transfected with siRNA of TAUT. TAUT Protein expression was measured by western blot and TAUT function with or without siRNA interference were then measured by [3H] taurine uptake examination. TRACP staining were applied for counting the number of osteoclasts.Results:M-CSF and RANKL induced bone marrow-derived macrophages expressed TAUT mRNA and TAUT protein. Taurine intervention could significantly inhibit osteoclastogenes. while TAUT siRNA relieved this effect.Conclusion:Taurine played important role in bone homeostasis by inhibiting osteoclastogenesis directly through TAUT. Taurine lost the ability to inhibit osteoclastogenesis when TAUT expression was blocked.