In vitro transfection of triple-point mutants of hypoxia-inducible factor 1 alpha into bone marrow mesenchymal stem cells

Hui Chun-ying; Xiao Hong-yan; He Xin-ling; Wang Wei

doi:10.3969/j.issn.2095-4344

Abstract：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 al kinds of cels and healing of injury is also promoted by vascular network. <br> OBJECTIVE:To study the effect of triple-point mutants of hypoxia-inducible factor1α (HIF1α) to promote angiogenesis after spinal cord injury in normoxic condition. <br> METHODS:Site-directed mutagenesis of 402, 564 and 803 amino acids in human HIF1α 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 HIF1α 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 cels. We observed transfection efficiency of virus by enhanced green fluorescent protein and to detect mRNA and protein expression of HIF1α gene in al transfection cels. We also detected protein expression of vascular endothelial growth factor acting as downstream angiogenesis gene of HIF1α in four groups by Western blot. <br> RESULTS AND CONCLUSION:Three adenoviral recombinants were successfuly 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 al of them were changed to alanine. The level ofHIF1α mRNA expression in both A group andB 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.