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Objective@# To investigate the effects of different glucose concentration on the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSC) in vivo@*Methods@#Cultured with basal medium containing different glucose concentrations, CCK-8 cell proliferation was detected at 1, 4, 7, 10 days. The osteogenic differentiation of human bone marrow mesenchymal stem cells was observed at 7 d, which was induced by osteogenic differentiation medium with different concentration of glucose. The expressions of alkaline phosphatase (ALP), osteocalcin (OC) and collagen type I (Col-1) gene were detected by real-time fluorescence quantitative PCR. Mineralized nodule formation was displayed by calciumalizarin red staining on the seventh day.@*Results @#10 mM glucose stimulated proliferation of hBMSC, while the higher (>30 mM) inhibited the proliferation (P < 0.05); Osteogenic induction can induce osteogenic differentiation of hBMSC, but the increase of glucose concentration will decrease the formation of mineralized nodules of hBMSC, inhibit the expression of osteogenic marker genes ALP, OC and Col-1 (P < 0.05).@*Conclusion@#The expression of Col-1, ALP and OC in osteoblast was down-regulated by high glucose, and the hBMSC proliferation was inhibited. At the same time, high glucose can reduce the osteogenic mineralization ability of stem cells and indirectly affect bone formation and metabolism.
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Objective To explore the safety of the human bone marrow‐derived mesenchymal stem cells (hBMSCs) after silencing of human leukocyte antigen A2 expression .Methods We divided the cells into three groups:normal cultured cells of the 8th passage served as control group , and hBMSCs after HLA A2 silencing expression of the 5th and 15th passage as experimental groups 1 and 2 ,respectively .The hBMSCs were recultured by sterile methods .The growth curve ,telomerase activation ,and expressions of P27 ,cyclin D2 and cyclin‐dependent kinase 4 (CDK4) were utilized to explore the safety of the hBMSCs induced by LV‐siRNA‐HLA A2 .The BMSCs were transplanted to the subcutaneous layer of nude mice .Tissue types were detected 24 weeks after transplantation . Results The cell curves had no obvious left or right shift in all the groups . The telomerease activation in experimental groups 1 and 2 did not significantly differ from those in control group . The expressions of anti‐oncogene P27 ,cyclin D2 and CDK4 had no obvious difference between the two experimental groups and control group , either . There was only ectopic osteogenesis 24 weeks after the BMSCs (HLA A2 gene silenced ) were transplanted to the subcutaneous layer of the nude mice .Conclusion There was no obvious evidence to support that hBMSCs had undergone change in safety after the silencing of HLA A 2 expression .
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BACKGROUND: Osteoporosis is one of the most serious side effects of long-term glucocorticoid therapy, but the mechanism of glucocorticoid-induced bone loss remains poorly defined. Glucocorticoid induces decreased bone formation and death of isolated segments of bone (osteonecrosis) suggesting that glucocorticoid excess may affect the birth or death rate of bone cells and thereby reduce their numbers. It has been known that reduction in bone formation is due to reduced proliferation in osteoblast precursor cells and reduced matrix synthesis in mature osteoblast. Here, we present evidence for dexamethasone-induced apoptosis on human bone marrow stromal cells (hBMSC). To understand the mechanism of glucocorticoid-induced osteoporosis, we investigated the effects of glucocorticoid on primary cultured hBMSC. METHEODS: Treatment with dexamethasone at the concentration of 10-9 M for 3~5 days significantly decreased cleavage tetrazolium salt WST-1 level/concentration by mitochondrial dehydrogenase in viable cells. Greater decrease was observed with higher concentration of dexamethasone (10-7 M, and 10-5 M). Apoptosis was measured by annexin V binding/propidium iodide using fluorescence-activated cell sorter (FACS) analysis and nuclear morphology stained with the fluorescence dye, Hoechst 33342. RESULTS: The level/concentration of apoptotic hBMSC (annexin V positive / PI negative) was increased with 10-9 M dexamethasone (1.2% to 5.3%) and further increased with 10-7 M, and 10-5 M concentration (11.7% and 12.5%, respectively). The same result was observed with Hoechst 33342 staining. CONCLUSION: These results indicate that glucocorticoid induces apoptosis on osteoblast precursor cell, hBMSC, and may contribute to decrease bone formation