Aims To investigate the effect of HIF-1a on MSC under hypoxia condition.
Materials and methods We transfected HIF-1a into MSC of P3 generation through liposome 2000, and observed the expression of green fluorescence protein in order to assess transfecting efficiency. G418 was used to screen stable transfected cells, and limited dilution method used for monoclone culture of screened cells. We identified the stable HIF-1a transfected MSC through the cell surface antigen testing. We compared the growth state among stable transfected MSC with HIF-1a, vacant plasmid transfected MSC and untransfected MSC under hypoxia condition, and the expression of HIF-1a mRNA, VEGF mRNA, HIF-1a protein and VEGF protein was tested.
Results pcDNA3.0-HIF-1a-eGFP can be successfully transfected into MSC mediated by liposome 2000, with efficiency of 21%. Stable monoclone of transfected MSC can be obtained by G418 screening and limited dilution method. Stable transfected MSCs still reserve the ability of differentiating to chondrocyte and lipocyte. MSCs transfected with pcDNA3.0-HIF-1a-eGFP had lower apoptosis (p<0.05), greater proliferation (p<0.05), and more expression of HIF-1a mRNA, VEGF mRNA, HIF-1a protein, VEGF protein than MSCs transfected with vacant plasmid pcDNA3.0- eGFP and untransfected ones under hypoxia condition.
Conclusions Stable transfected MSC with HIF-1a has a significant high expression of HIF-1a protein, HIF-1a mRNA, VEGF protein and VEGF mRNA under hypoxia condition. HIF-1a could reduce MSC apoptosis and enhance its proliferation under hypoxia condition.
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