title: [The study of osteogenic differentiation in canine bone marrow stromal cell induced by point mutant type of HIF-1α].
abstract: OBJECTIVE
  To evaluate osteogenesis in canine bone marrow mesenchymal stem cells(BMSCs) transduced by point mutant HIF-1α.
  
  
  METHODS
  Lenti-WT (wild type HIF-1α), Lenti-MT (mutant type HIF-1α), and Lenti-LacZ (the control group) were constructed with LR recombination system. And then Lenti-LacZ, Lenti-WT and Lenti-MT were used to transduce canine BMSCs. After transduction, total RNA and protein were extracted at 0 , 1 , 4 , 7, 14 d and 21 d, respectively. The mRNA and protein expression of osteogenic factors were detected by RT-PCR and Western blot in vitro under normoxic conditions.To further prove osteogenic differentiation of HIF-1α mediated BMSCs, the BMSCs were inoculated into 6-well plate (2×10(5)/well) and then the expression of calcium nodules was measured using Alizarin red staining(ARS) at 14 d and 21 d, respectively.
  
  
  RESULTS
  At MOI=9, the transduced efficiency of BMSCs was up to 90%. After target gene being transduced to BMSCs, the mRNA and protein expression of osteogenic factors were significantly up-regulated at 4d, reached peak at 14-21 d and maintained a state of high expression (P<0.05). ARS results showed that the target gene can induce BMSCs to the osteogenic differentiation in normoxic conditions.
  
  
  CONCLUSIONS
  Under normoxic conditions in vitro, mutant HIF-1α can stabilize the expression and maintain a high level of activity. Lenti-MT can significantly improve the osteogenic activity of canine BMSCs.
authors.affiliation: Department of Stomatology, Bay Hospital of Fengxian District, Shanghai, China. yujianyingyy@163.com
authors.forename: Jian-ying
authors.initials: JY
authors.lastname: Yu
authors.forename: Si-heng
authors.initials: SH
authors.lastname: Zhu
authors.forename: Duo-hong
authors.initials: DH
authors.lastname: Zou
mesh_terms: D001854:Bone Marrow Cells; D002454:Cell Differentiation; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D059630:Mesenchymal Stem Cells; D010012:Osteogenesis
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit