Effects Of Ginsenoside Rg1on The Proliferation And Osteoblast Differentiation Of Human Periodontal Ligament Stem Cells In Vitro

Objective:This experiment was performed to examine the effect of of ginsenoside Rgl on the proliferation and osteoblast differentiation of human periodontal ligament stem cells. human periodontal ligament cells (hPDLSCs) were successfully primary cultured in vitro, then we treated with different concentrations of ginsenoside Rgl of mineralized induced medium. The effects of different concentrations of ginsenoside Rgl(10-8,10-7,10-7,10-15,10-4mol/L) were evaluated by the methylthiazolytetrazolium (MTT), ALP activity, mineralized nodules staining and osteogenesis related genes(Runx2, Collagen I,OPN, OCN),in order to reveal its mechanism and understand the pharmacological of ginsenoside Rgl on hPDLSCs. So this study provided a theoretical foundation of developing ginsenoside Rgl for periodontal regeneration tissue repair and treatment of periodontal disease.Method:1.Normal impacted third molars of healthy individuals were extracted, and PDLSCs were separated and cultured as previously reported. Then use microscope to observe the morphological features, clone forming experiment testing the clone forming rate,2.By detecting the expression of immune molecules related expert witnesses the periodontal ligament stem cell phenotype,3.The multiple differentiations of PDLSCs, including the osteogenetic differentiation and differentiation into fat,4.Under the condition of mineralized induced concentration for10-8,10-7,10-6,10-5,10-4mol/L ginsenoside Rgl and periodontal ligament stem cells Id,2d,3d,4d,5d, using methyl thiazolyl blue tetrazolium bromide (determined by MTT) evaluation ginsenoside Rgl on the influence of the periodontal ligament stem cells proliferation,5.Under the condition of mineralized induced the concentrations of10-8,10-7,10-6,10-5mol/L ginsenoside Rgland periodontal ligament stem cells in3d,5d,7d, testing the ALP activity,6.Under the condition of osteogenesis induced concentration for10-8,10-7,10-6,10-5mol/L ginsenoside Rgl14d and the periodontal ligament stem cells, alizarin red S staining, observed calcium salt deposition,7. Real-time fluorescence quantitative PCR detect Runx2, Collagen I, OPN, OCN expression.Results:1.PDLSCs form a radial and spiral shape,which also have the strong ability of clone formation.2.Positive expression between markers and CD146and Stro-1.3.Osteogenic differentiation showed that the wells were almost completely covered with mineralized deposits, as revealed in using alizarin red staining after14days. Under chondrogenic culture conditions,14days later with regard to adipogenic differentiation, cells showed a larger number of clusters of lipid droplets after21days of adipogenic differentiation.4.At ginsenoside Rgl concentrations of10-8to10-4mol/L, OD values increased substantially from days2to5and were higher than that of the blank control (P<0.05). OD values increased with increasing Rgl concentration, and a dose-dependent effect was showed. On the other hand, in the group of10-4mol/L ginsenoside Rgl decreased rapidly, indicating that the of ginsenoside Rgl this concentration was seriously poisonous to cell growth. Thus, in the following experiment, the10-4mol/L group was deleted.5.At ginsenoside Rgl concentrations of10-7to10-5mol/L, ALP expression increased substantially from days3to7and was higher than that of the blank control (P<0.05). ALP expression increased with increasing ginsenoside Rgl concentration, and a dose-dependent effect was showed. On the other hand, at different time point,10-5mol/L ginsenoside Rgl showed the highest expression of ALP in all the ginsenoside Rgl groups (P<0.05).6.PDLSCs were cultured through osteogenic differentiation medium with the different factors for14days and then the intensity of any calcium deposits was determined by staining with alizarin red S and in a quantitative biochemical colorimetric method. Cells were treated with10-8to10-5mol/L, showed slightly enhanced calcium nodule formation, especially10"5mol/L.7.The RT-PCR result for the detection of osteoblastic differentiation markers showed that all osteogenic differentiation genes’ expressions were increased in ginsenoside Rgl solutions as compared to the control groups.The dose-dependent ginsenoside Rg1(10-8mol/L-10-5mol/L) increased the expression of these osteogenic differentiation markers (Runx2,Collagen I,OPN, OCN).Conclusion:1.To isolate human periodontal ligament stem cells in vitro and purify them by limiting dilution of cloning assay, laid a good foundation for further study.2.The periodontal ligament stem cells have the strong ability of clone formation; and high expression CD146and Stro-1, which explain the source of ectomesenchymal;Under specific conditions, they could differentiate to the osteoblast and adipocyte lineages.3.In vitro culture condition, the detection of10-8mol/L,10-7mol/L,10-6mol/L,10-5mol/L groups of ginsenoside Rgl can promote the proliferation of periodontal ligament stem cells, and10"5mol/L group of ginsenoside Rgl promote proliferation most obviously, and the detection of10-4mol/L group of ginsenoside Rg1inhibit the growth of the periodontal ligament stem cells.4.Human periodontal ligament stem cells under the conditions of osteogenesis induce with10"8mol/L,10-7mol/L,10-6mol/L,10-5mol/L groups of ginsenoside Rgl,the group of10-5mol/L showed the highest expression,and the expression of bone-building genes Runx2, Collagen I, OPN, OCN were raised.