Mutants of hypoxia-inducible factor-1αaccelerate in vivo angiogenesis in bone defect region
10.7652/jdyxb201504006
- VernacularTitle:突变型低氧诱导因子1α加速骨缺损部位新血管生成的实验观察
- Author:
Hao WANG
;
Chen LI
;
Yuzhong GAO
- Publication Type:Journal Article
- Keywords:
hypoxia-inducible factor 1α;
mutagenesis;
recombinant adenovirus vector;
bone marrow mesenchy-mal stem cell;
angiogenesis;
bone defect region
- From:
Journal of Xi'an Jiaotong University(Medical Sciences)
2015;(4):455-461
- CountryChina
- Language:Chinese
-
Abstract:
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 ).There was no significant difference between Group A and Group B group or between 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);however,there was no significant difference among groups B,C and D (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.
