Regulation of Sox-9 and hypoxia-inducible factor-1alpha in adipose-derived mesenchymal stem cells

Zhang Chuan-hui; Li Jian-jun; Yang Jun

doi:10.3969/j.issn.2095-4344.2016.45.012

Abstract：BACKGROUND:Gene transfection of adipose-derived mesenchymal stem cel s (ADSCs) can stably synthesize seed cel s that secrete specific cytokines, thereby achieving long-term stable proliferation, differentiation and chondrogenic differentiation of seed cel s. <br> OBJECTIVE:To observe the effect of insulin-like growth factor-1 (IGF-1) gene transfection on the expression of Sox-9 and hypoxia-inducible factor-1alpha (HIF-1α) in rabbit ADSCs. <br> METHODS:ADSCs were harvested from the posterior subcutaneous adipose tissue from Adult New Zealand white rabbits, then the cel s were identified by immune fluorescent assay. After being transfected with pcDNA3.1-IGF-1, the cel s were selected with G418. RT-PCR and western blot were used to analyze the expression level of HIF-1αin normal ADSCs, pcDNA3.1-transfected ADSCs, and pcDNA3.1-IGF-1-transfected ADSCs. The growth curve of cel s was measured by MTT, and meanwhile, proliferation efficiency of cel s was measured by counting CM-DiL-labeled cel s. Immune fluorescent assay was used to analyze the expression of HIF-1αand Sox-9. And the content of glycosaminoglycan in each group was determined by using dimethylmethylene blue dye-binding assay. <br> RESULTS AND CONCLUSION:The stably expressed pcDNA3.1-IGF-1 cel lines were successful y established and showed stable expression of IGF-1 at mRNA and protein levels. MTT and CM-Dil fluorescence results suggested that IGF-1-transfected cel s displayed stronger proliferation ability and efficiency, and these cel s showed a positive expression of HIF-1αand Sox-9 as wel as higher glycosaminoglycan content than the other two groups (P<0.01). Al these findings indicate that IGF-1 gene transfection can effectively promote the proliferation of ADSCs, and promote the positive expression of Sox-9 and HIF-1α. And the secretion of chondral extracel ular matrix such as glycosaminoglycan is also significantly improved.