Effects Of Glucose And O-GlcNAcylation On HBMSCs

BackgroundMesenchymal stem cells derives from the mesoderm.Bone marrow mesenchymal stem cells can convert between germ layers under appropriate conditions.Mesenchymal stem cells can differentiate into chondrocytes,adipocytes,osteoblasts and even blood vessels under inducing conditions.Mesenchymal stem cells are so important as seed cells in the field of tissue engineering,due to it's high proliferative capability in vitro.Glucose is an important metabolite for most cells.Glucose plays important roles in cell proliferation,apoptosis,senescence and differentiation in microenvironment.Hexosamine synthetic pathway may act as a sensor of cell status of nutrition.Glucose could regulate the level of O-GlcNAcylation of protein in cells through hexosamine synthetic pathway.O-GlcNAcylation is an important post-translational modification of proteins.And it works the same as phosphorylation and plays an important role in cellular living.It has been found that many nuclear proteins,cytoplasmic proteins and mitochondrial proteins can be modified by O-GlcNAcylation.O-GlcNAcylation intracellular is highly relevant to proliferation,apoptosis,senescence and differentiation of cells.So it is valuable to explore the effect of glucose on the hBMSCs and what roles does O-GlcNAcylation play.Methods1.Group of experiments:Normal-glucose group(glucose 5.5mmol / L),high-glucose group(glucose 25 mmol / L),normal-glucose+O-GlcNAcylation group(glucose 5.5mmol / L,Thiamet-G 1.0mol / L),high-glucose+O-GlcNAcylation group(glucose 25 mmol / L,Thiamet-G 1.0mol / L).2.Effect of glucose on O-GlcNAcylation.3.Effect of glucose and O-GlcNAcylation on proliferation of hBMSCs.WST-1 cell proliferation and cytotoxicity assay kits evaluates proliferation of each group.4.Effect of glucose and O-GlcNAcylation on cell cycle of hBMSCs.Flow cytometry evaluates cell cycle of hBMSCs,and RT-PCR evaluates cyclin D1 expression.5.Effect of glucose and O-GlcNAcylation on apoptosis of hBMSCs.Flow cytometry evaluates apoptosis of hBMSCs,and RT-PCR evaluates the expression caspase-3.6.Effect of glucose and O-GlcNAcylation on senescence of hBMSCs.?-galactosidase staining evaluates senescence of hBMSCs.7.Effect of glucose and O-GlcNAcylation on chondrogenic differentiation of hBMSCs.Alcian blue staining analyzes chondrogenic differentiation of hBMSCs.RT-PCR evaluates chondrogenic differentiation markers,including Sox9,Col ?,AGN.Western blot evaluates the expression of PKC,p-PKC,and TGF-?R?.Results1.High concentration of glucose in microenvironment can increase O-GlcNAcylation of proteins in hBMSCs.2.High concentration of glucose and the high O-GlcNAcylation of proteins inhibite the proliferation of hBMSCs.3.High concentration of glucose and high O-GlcNAcylation of proteins block the transition of G1/S phase of hBMSCs.High glucose concentration and high O-GlcNAcylation of proteins inhibite the expression of Cyclin D1.4.High concentration of glucose and high O-GlcNAcylation of proteins increase the apoptosis of hBMSCs.High concentration of glucose and high O-GlcNAcylation of proteins also contribute to the increase of caspase-3.5.High concentration of glucose and O-GlcNAcylation of proteins increase the senescence of hBMSCs.6.High concentration of glucose and high O-GlcNAcylation of proteins inhibite the expression of differentiation markers such as Sox9,Col ?,AGN,which means them inhibite hBMSCs chondrogenic differentiation.7.High level O-GlcNAcylation of intracellular proteins inhibites the phosphorylation and activity of PKC,then leading to the lower expression of TGF-?R?.Conclusion1.In this study,we demonstrated that high concentration of glucose can increase the O-GlcNAcylation of proteins in hBMSCs.2.This study demonstrated that the high concentration of glucose inhibited the proliferation of hBMSCs,inhibited the G1/S phase transition,promoted its apoptosis and senescence.At the same time,the O-GlcNAcylation of proteins and high concentration of glucose have the same effect,but the extent of the latter is higher than O-GlcNAcylation of proteins.Glucose may affect the intracellular O-GlcNAcylation level through HBP,so the increase of O-GlcNAcylation of proteins may be one of the mechanisms of high concentration glucose affects hBMSCs.3.In this study,we demonstrated that the high concentration of glucose and O-GlcNAcylation inhibites the differentiation of hBMSCs into cartilage.And high level of O-GlcNAcylation inhibites PKC phosphorylation and activity,which causes the decrease of the expression of TGF-?R?.