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Objective To investigate the functions of triple point-mutants of hypoxia-inducible factor 1α(HIF1α)in angiogenesis in bone defect region under normoxic conditions.Methods Triple point-mutations (the 402,564 and 803 amino acids)in HIF1α coding sequence (CDS)were induced.The triple mutant of HIF1α(402/564/803)was inserted into the adenovirus pAdEasy-1 system to complete viral packaging and titer measurements. The wild-type HIF1α gene and the empty adenovirus vector were packaged in the same way.For the in vitro experiment,the rabbit bone marrow mesenchymal stem cells (MSCs)were divided into four experimental groups:A,MSCs infected with viral solution containing mutant HIF1α;B,MSCs infected with viral solution containing wild-type HIF1α;C,MSCs infected with viral solution without any HIF1α;and D,MSCs without viral infection. The efficiency of infection was observed by the expression of human renilla reniformis green fluorescent protein (hrGFP).The expression levels of HIF1α mRNA and protein in infected cells in each experimental group were measured.For the in vivo experiment,the MSCs were divided into the same four groups and infected with the virus solutions from each group and cultured under normoxic conditions.At 72h after the infection,the MSCs were used as seed cells and transplanted into Apatite-wollastonite magnetic bioactive glass-ceramic (AW MGC)vector to construct artificial tissue-engineering scaffolds,and then the scaffolds were implanted into the in vivo rabbit radial bone defect model.The animals from each group were sacrificed 8 weeks after the surgery and the tissues from the implantation region were harvested for evaluation of angiogenesis.Results The 402,564 and 803 amino acids in CDS area were point mutated into alanine;three types of recombinant adenovirus were successfully constructed, packaged and characterized.The expression levels of HIF1αmRNA were significantly higher in Group A and Group B than in Group C and Group D (P 0.05 ).The HIF1α protein expression in Group A was significantly higher than that in the other three groups (P 0.05).There was prominent angiogenesis in bone defect region in Group A,but not in the other three groups.Conclusion ① Triple point-mutants of HIF1αefficiently expressed functional proteins under normoxic conditions.② Triple point-mutants of HIF1αeffectively promoted in vivo angiogenesis in bone defect region.
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BACKGROUND:Lipopexia induced by glucocorticoid is fat precipitation in marrow due to abnormal lipomatabolism,or differentiation of cells resulting from hormone-affected bone marrow mesenchymal cells.The precise generating procedure remains unclear.OBJECTIVE:To Jnvastigate effects of vadous concentrations of dexamethasone on adipogenic differentiation of bone marrow mesenchymal cells.DESIGN,TIME AND SETTING:The observational study was performed at the Department of Biochemistry,China Medical University from January 2007 to January 2009.MATERIALS:A total of 80 Sprague-Dawley rats aged 3 or 4 weeks,weighing (110±10) g,of both genders,were used in this study.METHODS:Rat bone marrow mesenchymal cells were isolated and subcultured.Bone marrow masenchymal cells at the third passage were used as samples.MAIN OUTCOME MEASURES:Cellular morphologic changes after treatment of 10~(-7) mol/L dexamethasone were observed under an inverted microscope,as well as at 3,7,14 and 21 days under normal culture condition.Morphological changes of bone marrow mesenchymal cells were observed following alkaline phosphatase and Sudan Ⅲ staining.Alkaline phosphatase activity was measured using phenol reagent.Effects of dexamethasone on cell proliferation were measured using MTT assay.RESULTS:Following 24 hours of incubation,a few adherent cells were found in the bottom of the culture flask,showing spindle shape.With prolonged time,adherent cells became more,presenting radiated shape.Following passage,cells distributed uniformly,showing typical fibroblast-shape.Following dexamethasone stimulation,cells changed from spindle-shape into polygonal or irregular shape.In the late phase,with increased concentration and prolonged time,cell colonies disappeared;cells adhered,and died.In normal cultured cells,no or a few orange particles were found.In dexamethasone-cultured cells,with increased hormone concentration and prolonged time,Sundan Ⅲ stained particles increased.At 21 days,alkaline phosphatase activity under normal culture was separately 1.57-,4.49-and 5.0-fold of 10~(-8),10~(-7),10~(-6) mol/L dexamethasone group.At 7,14 and 21 days,alkaline phosphatase activity under normal culture was separately 2.93-,3.80-and 4.39-fold of 10~(-7) mol/L dexamethasone group (P ＜ 0.05).High concentration of dexamethasone had significant inhibitory effects on cell proliferation activity.Significant difference was detected as compared 10~(-7) mol/L and 10~(-6) mol/L to other groups (P ＜ 0.05).CONCLUSION:High-dose dexamethasone enhances adipogenic and suppresses osteoblastic differentiation of bone marrow mesenchymal cells.The effect will increase along with the increasing content and prolonged duration of demamethasone stimulation.