Mutant HIF1α Modified Bone Marrow Mesenchymal Stem Cells Ameliorate Cerebral Ischemia

ObjectiveHIF-1α plays stimulatory roles in the revascularization in ischemic area of cerebral ischemia. However, the hydroxylation of proline at 564 and asparagine at 803 in the HIF1α coding sequence facilitated the degradation of HIF and inhibited the transcription activity of HIF-1α promoter under normoxic conditions and confined the pro-angiogenic efficacy of HIF-1α.MethodsIn this study, HIF-1α mutant containing P564 A and N803 A was constructed by site-directed mutagenesis on the basis of p Ad Easy-1-HIF1α and packaged in HEK293 A and using the method of end-point virus dilution drops to complete the mutant HIF1α construction of adenovirus vector. Rat bone marrow mesenchymal stem cells(BMSCs) were infected with adenoviral particles containing HIF1α mutant at multiplicity of infection. The HIF1α m RNA and protein levels under hypoxia and normoxic conditions were compared using RT-PCR and western blot. To explore the therapeutic effect of mutant HIF1α on the cerebral ischemia, BMSCs overexpressing mutant HIF1α were transplanted in the rat middle cerebral artery occlusion model(MCAO). The motor function and cerebral infarction size were evaluated using modified neurological severity score(m NSS) and triphenyltetrazolium chloride(TTC) staining within four weeks after MCAO. VEGF protein expression was detected by Western Blot. Microvessel density and angiogenesis were detected by immunohistochemistry to evaluate the recovery of the brain ischemia.Results1、We successfully point-mutated 564 and 803 to construct mutant HIF1α,and it is same to positive Wild type HIF1α and negative control virus vector. HIF1α mutant containing P564 A and N803 A could be expressed under normoxic condition. The transfected HEK293 A cells were observed under a fluorescence microscope. The images showed cytopathic effects(CPE): cell soma swollen and part of the cells falling off from the wall.2、 HIF1α and mutant HIF1α transfected BMSCs showed strong expression of green florescent protein indicating that mutant HIF1α can be expressed at high level under nomorxic conditions.3、 RT-PCR showed that the expression of mutant HIF1α m RNA could be detected under normoxic conditions. Western blot analysis revealed that the mutant HIF1α protein was only detected under normoxic conditions.4 、 Transplantation of BMSCs stably expressing mutant HIF1α significantly improved motor function, reduced cerebral infarction, and increased VEGF protein expression neovascularization at days 7, 14 and 28(P<0.05).ConclusionsHIF1α mutant containing P564 A and N803 A may be a potential target for the treatment of the cerebral ischemia.