Effects Of High Glucose Microenvironments On Proliferation And Migration Of BMSCs Through Cyclin D1 And CXCR-4 Mediated By GSK3β

With the popularity of planting technology, high failure rate in diabetes patients have been taken seriously. Diabetes is a metabolic diseases of hyperglycemia. Numerous studies have confirmed that diabetes can cause abnormal bone metabolism and poor quality bone formation[l], the poor quality of the mandible and dental implant are closely related.BMSCs plays an important role in bone metabolism. It has been confirmed that high glucose could affect the physiological functions and the quality of osteogenesis of BMSCs.Glycogen synthase kinase 3β (GSK3β) is a serine/threonine protein kinase which has a variety of physiological functions, GSK3P is an important regulatory protein of glucose metabolism, it plays an important role in the change in the regulation of glucose levels and diabetes-related indicators.GSK3β can regulate β-Catenin phosphorylation and stability, and Cyclin D1 is not only a target gene of P-catenin/LEF-1 pathway downstream, but also a factor closely related to cell proliferation. The research has also demonstrated inhibition of GSK3β can promote mesenchymal stem cell migration by increasing the expression of CXCR-4.So GSK3β is an important factor which is closely related to the Cyclin D1 and CXCR-4.The microenvironment of stem cells is the factors which is affect the physiological functions of stem cells such as proliferation, differentiation, etc. In this study, we cultured the BMSCs in high glucose microenvironment, and tested the proliferation and migration ability of BMSCs, and explore the mechanism of the affection on proliferation and migration by GSK3β,Cyclin D1 and CXCR-4. Provide a theoretical basis for improve the success rate of implant in diabetes.PartⅠ:Objective:To isolate the BMSCs from SD rats and identify the cell phenotype.To test the physiological functions such as proliferation, colony-forming ability and adipogenic and osteogenic differentiation potential.Methods:We isolated the BMSCs from SD rats (3 weeks age) and cultured. We used FCM to test the expression of CD90, CD105, CD34 and CD45. We tested the proliferation of BMSCs by MTT and investigated the colony-forming ability and osteoblastic/adipogenic differentiation potential.Results:The BMSCs were morphology uniform, and positive for CD90 and CD 105, negative for CD34 and CD45.The cells were at the stage of logarithmic growth phase in day 3 and at the stage of plateau in day 6.The cells had the ability of colony-forming,the cells have the ability of osteoblastic/adipogenic differentiation potential. Conclusion:We successfully isolated the BMSCs from SD rats, and the expression of CD markers were conformed to the stem cell markers. The cells had the potential ability of proliferation, colony-forming and differentiation.Part Ⅱ:Objective:To explore the effection of high glucose microenvironment on proliferation of BMSCs by Cyclin D1.Methods:We test the proliferation ability of BMSCs by MTT and cell cycle in different concentrations of glucose (5.5mM,16.5mM). We test the mRNA expression of Cyclin D1 by RT-PCR and protein expression of Cyclin D1 and CDK4 by westrn blot. Results:The result of MTT revealed high glucose could inhibit the proliferation and progression from the G1 phase to the S-phas of BMSCs. And high glucose also could inhibit the expression of Cyclin D1 and CDK4.Conclusion:High glucose could affect the proliferation of BMSCs by inhibiting the expression of Cyclin Dl in BMSCs.Part III:Objective:To explore the effection of high glucose microenvironment on migration of BMSCs by CXCR-4Methods:We tested the migration of BMSCs with SDF-1 and AMD3100 in different concentrations of glucose (5.5mM,16.5mM) by transwell. We tested the expression of CXCR-4 by RT-PCR and western blot.Results:Our data revealed that SDF-1 greatly increased BMSCs migration, while the CXCR-4 inhibitor AMD3100 nhibited this increasing trend in the presence of 5.5 mM and 16.5 mM glucose.We also found that high glucose microenvironments reduced BMSCs migration in the presence of SDF-1.Conclusion:high glucose microenvironments reduce BMSCs migration through SDF-1/CXCR-4 axis by decreasing CXCR-4 expression Part III:Objective:To explore the effection of high glucose microenvironments on proliferation and migration of BMSCs by GSK3β, Cyclin D1 and CXCR-4.Methods:We tested the expression of GSK3β and β-Catenin of BMSCs with LiCl in different concentrations of glucose by western blot. We tested the mRNA expression of LEF-1, Cyclin D1 and CXCR-4 of BMSCs with LiC1 in different concentrations of glucose by RT-PCR. We tested the proliferation of BMSCs with LiCl in different concentrations of glucose by MTT and cell-cycle. We tested the migration of BMSCs under the condition of SDF-1 with LiCl in different concentrations of glucose by transwell.Results:High glucose could activate the GSK3β but inhibit β-catenin, LEF-1 and downstream factor Cyclin D1. GSK3β inhibition could promote the expression of P-catenin, LEF-1 and Cyclin D1. High glucose also could inhibit the expression of CXCR-4,GSK3p inhibition could promote the expression of CXCR-4. High glucose could inhibit the proliferation and migration of BMSCs. GSK3β inhibition could promote the proliferation and migration. High glucose could inhibit the progression from the G1 phase to the S-phas.GSK3β inhibition could promote the progression from the G1 phase to the S-phas.Conclusion:High glucose could affect the proliferation and migration of BMSCs by GSK3P, which is a regulator of Cyclin Dl and CXCR-4.