期刊
CELL BIOCHEMISTRY AND BIOPHYSICS
卷 67, 期 2, 页码 557-566出版社
HUMANA PRESS INC
DOI: 10.1007/s12013-013-9541-8
关键词
Hypoxia-inducible factor 1 alpha; Mutagenesis; Recombinant adenovirus vector; Bone marrow mesenchymal stem cells; Angiogenesis
资金
- Liaoning Affiliated Hospital Youth Science and Technology Startup Fund [FY2012-04]
To investigate the functions of triple point-mutants of hypoxia-inducible factor 1 alpha (HIF1 alpha) in angiogenesis in bone defect regions under normoxic conditions. 1. Triple point-mutations (in amino acids 402, 564, and 803) in the HIF1 alpha coding sequence (CDS) were induced by polymerase chain reaction. The triple mutant HIF1 alpha (402/564/803) was inserted into the adenovirus pAdEasy-1 system for complete viral packaging and titer measurements. 2. For the in vitro experiment, rabbit bone marrow mesenchymal stem cells (MSCs) were divided into four experimental groups. The efficiency of infection was observed by the expression of human renilla reniformis green fluorescent protein (hrGFP). The HIF1 alpha mRNA, protein and VEGF protein expression levels in infected cells in each experimental group were measured. 3. As in the in vivo experiment, the MSCs were divided into four groups and infected with the viral solutions from each complementary in vitro group and cultured under normoxic conditions. The MSCs were used as seed cells and transplanted into an apatite-wollastonite magnetic bioactive glass-ceramic (AW MGC) vector to construct artificial tissue-engineering scaffolds that were then implanted into the in vivo rabbit radial bone defect model. The animals from each group were killed 8 weeks after the surgery, and the tissues from the implantation region were harvested for the evaluation of the angiogenesis. 1. 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. 2. The expression levels of HIF1 alpha mRNA in A and B groups were significantly higher than those in the C and D groups (P < 0.05). The HIF1 alpha and VEGF protein expression levels in A group were significantly higher than those in the other three groups (P < 0.05). 3. There was prominent angiogenesis in bone defect regions in group A animals. 1. Triple point-mutants of HIF1 alpha efficiently expressed functional proteins under normoxic conditions. 2. Triple point-mutants HIF1 alpha effectively promoted in vivo angiogenesis in bone defect regions.
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