Recombinant adenovirus-mediated hypoxia inducible factor 1alpha mutant promotes proliferation of adipose-derived mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND:To improve the survival rate of transplanted tissue, most scholars focus on cel therapy, particularly cel-assisted fat grafting. OBJECTIVE:To analyze the effect of recombinant adenovirus-mediated hypoxia inducible factor 1alpha (HIF1α) mutant on survival rate of transplanted fat particles through transfection of adipose-derived mesenchymal stem cels. METHODS:Recombinant adenovirus-mediatedtriple-mutantHIF1αwas inserted into an adenovirus pAdEasy-1 system, folowed by viral packaging and titer determination. Human adipose-derived mesenchymal stem cels were cultured, passaged and identified, and subsequently transfected with three kinds of viruses and blank vector (experimental group with transfection of the triple mutantof HIF1α;positive control group; negative control group; blank control group). Transfection efficiency was determined using enhanced green fluorescent protein labeling. Additionaly, MTT assay was used to detect cel proliferation. RESULTS AND CONCLUSION:The recombinant adenovirus was successfuly constructed and packaged in line with transfection requirements. Moreover, adipose-derived mesenchymal stem cels were successfuly identified by adipogenic, osteogenic and chondrogenic induction and could be used asseed cels for subsequent experiments. RT-PCR results showed that HIF1α mRNA expression in the experimental group and positive control group was significantly higher than that in the other two groups (P< 0.05). Western blot analysis showed that the relative absorbance value in the experimental group was significantly higher than that in the other three groups (P< 0.05). A significant increase in the cel proliferation was found in the experimental group, significantly different from the other three groups (P<0.05). Therefore, our findings indicate that transfection of adenovirus-mediated triple-mutant HIF1α not only can sustain the expression of target protein in transfected adipose-derived mesenchymal stem cels under normoxic conditions, but also can promote the proliferation of transfected adipose-derived mesenchymal stem cels.