SymbolIn vitrotransfection of triple-point mutants of hypoxia-inducible factor 1 alpha into bone marrow mesenchymal stem cells

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BACKGROUND:The incidence of spinal cord injury is increasing year by year in China so that the construction of effective vascular network in local injury as soon as possible is the guarantee of metabolism and nutritional support to differentiation of all kinds of cells and healing of injury is also promoted by vascular network.OBJECTIVE:To study the effect of triple-point mutants of hypoxia-inducible factor 1 α (HIF1α) to promote angiogenesis after spinal cord injury in normoxic condition.METHODS:Site-directed mutagenesis of 402, 564 and 803 amino acids in human HIF1 α coding sequence area was completed by PCR, and the adenovirus pAdEasy-1 system was recombined with post-mutation HIF1α gene. Packaging viral and titration determination of experimental group was completed and the same was done to non mutation group and control virus group. The future experiment was continued with three virus groups and blank group (A group: including mutation HIF1 α gene virus liquid; B group: including non mutation HIF1α gene virus liquid; C group: including control virus liquid; D group: blank group). Then, virus liquid was transferred into rat bone marrow mesenchymal stem cells. We observed transfection efficiency of virus by enhanced green fluorescent protein and to detect mRNA and protein expression of HIF1 α gene in all transfection cells. We also detected protein expression of vascular endothelial growth factor acting as downstream angiogenesis gene of HIF1 α in four groups by Western blot.RESULTS AND CONCLUSION:Three adenoviral recombinants were successfully constructed and the packaging and identification were accomplished. The site-directed mutations of 402, 564 and 803 amino acids in coding sequence area were successful and all of them were changed to alanine. The level of HIF1 α mRNA expression in both A group and B group were significantly higher than that in the C group and D group (P < 0.05). The expression levels of HIF1α and vascular endothelial growth factor proteins in A group was significantly higher than those in the other three groups (P < 0.05). These findings indicate that the HIF1α gene largely and effectively express in normoxic condition after triple-point mutation and the high-efficiency expression of vascular endothelial growth factor which is a downstream angiogenesis gene of HIF1α is promoted so that it is maybe a new therapeutic way of angiogenesis in the treatment of spinal cord injury.Funding:the Funded Project of the Social Development Department of Liaoning Science and Technology Bureau, No. 2012408002

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