| Objective:Type 2 diabetes,as a common chronic metabolic disease,increases the risk of fracture and interferes with the healing of bone tissue,especially affecting bone formation metabolism.Bone marrow mesenchymal stem cellsare important cells in the process of osteogenic metabolism,and can be used as seed cells in bone regenerative medicine tissue engineering,which has certain application prospects.Previous studies have confirmed that circadian genes interact with type 2 diabetes,and that circadian genes are involved in the regulation of a wide range of physiological activities in cells.Therefore,this study investigated the osteogenic differentiation ability and circadian gene expression of bone marrow mesenchymal stem cells by establishing a rat model of type 2 diabetes,and explored that the circadian gene Clock in the microenvironment of type 2 diabetes affects the function of mesenchymal stem cells.The regulation mechanism of bone differentiation provides a basis for prevention and treatment of bone damage caused by type 2 diabetes.Methods:(1)The rat model of type 2 diabetes was established and screened using Goto-Kakizaki rats,and Wistar rats were used as a control group.The mandible was separated and analyzed by morphometric scanning using Micro-CT.The bone marrow mesenchymal stem cells within the mandible were extracted and their purification and culture were conducted in vitro.(2)The proliferation and apoptosis of the two cell groups were evaluated by colony formation assay,flow cytometry,cell cycle and apoptosis assay,and CCK-8 assay.The two groups of cells were subjected to alkaline phosphatase staining 7 days after osteogenic induction,while alizarin red staining was performed 21 days after osteogenic induction.qRT-PCR and Western blot were used to detect the changes of circadian gene Clock,proinflammatory factors and apoptosis marker genes,osteogenic related genes and NF-κB classical signaling pathway markers expression.The luciferase assay was used to detect the changes of NF-κB classical signaling pathway activity in thetwo groups before and after osteogenic induction.(3)Constructed of lentiviral vector,packagedcircadian gene Clock-overexpressed lentivirus,transfected into diabetic group bone marrow mesenchymal stem cells.qRT-PCR was used to detect the changes of circadian gene Clock,osteogenic related genes and NF-κB classical signaling pathway markers in diabetic transfection group and fluorescence group,non-transfected diabeticgroup and Wistar control group.The luciferase assay was used to detect the changes of NF-κB classical signaling pathway activity in each group before and after osteogenic induction.Results:(1)The Goto-Kakizaki group of type 2 diabetes showed significantly higher fasting blood glucose values,fasting insulin values and glucose tolerance test values,as well as significantly lower body weight values.The bone morphometric parameters of the mandibular group in the diabetic group included bone mineral density,trabecular thickness,trabecular bone spacing,and bone volume fraction,which were not significantly different from the control mandible,indicating that there is no osteoporosis-like change in the mandible in the microenvironment of type 2 diabetes in the short term.(2)The number of cloned bone marrow mesenchymal stem cells in the diabetic group,the percentage of cells in S phase and G2 phase,and the absorbance value of CCK-8 were significantly lower than those in the control group,while the percentage of cells in G1 phase,early apoptotic rate and late apoptosis were significantly higher than that of the control group,indicating that the microenvironment of type 2 diabetes promoted the apoptotic activity of bone marrow mesenchymal stem cells and inhibited its proliferative activity.Compared with the control group,alkaline phosphatase staining and alizarin red staining of bone marrow mesenchymal stem cells in diabetic group were shallow and sparse,and its expression levels of osteogenic markers including OPN,Runx2,OSX and ALP were lower than those of the control group,indicating that the microenvironment of type 2 diabetes inhibited the osteogenic differentiation of bone marrow mesenchymal stem cells.The expression of the circadian gene Clock and proinflammatory factors including IL-1β,TNF-α,IL-6,apoptosis marker genes such as caspase-3 and caspase-8,and NF-κB classical signaling pathway marker genesp65 and p-IKK a were significantly increased before and after osteogeneic differentiation.And the dual luciferase assay showed that the NF-κB classical signaling pathway activity of bone marrow mesenchymal stem cells in diabetic group wassignificantly higher than that in the control group before and after osteogenic differentiation.(3)The expression level of the circadian gene Clock of bone marrow mesenchymal stem cells in the diabetic transfection group was significantly higher than that in the other three groups.And the expression level of NF-κB classical signaling pathway marker genesp65 and p-IKK a in the transfected group was significantly higher than those in the other three groups,and the osteogenesis-related marker genes Runx2 and ALP were significantly lower than those in the other three groups.The dual luciferase assay showed that the NF-κB classical signaling pathway activity of bone marrow mesenchymal stem cells in the diabetic transfection group was significantly higher than that in the other three groups.Conclusions:(1)There is no osteoporosis-like change in the mandible in the microenvironment of type 2 diabetes in the short term.(2)The proliferation activity and osteogenic differentiation ability of bone marrow mesenchymal stem cells in the microenvironment of type 2 diabetes were decreased.(3)In the microenvironment of type 2 diabetes,the expression of the circadian gene Clock of bone marrow mesenchymal stem cells increased,up-regulated the activity of NF-κB classical signaling pathway,and participated in the inhibition of osteogenic differentiation ability of cells.(4)The circadian gene Clock may become a regulatory target in tissue engineering of bone regenerative medicine. |
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