ABSTRACT
BACKGROUND: A large number of studies have confirmed that hypoxia can promote the proliferation and differentiation of stem cells, but the pathway by which it plays its role remains unclear. OBJECTIVE: To investigate the effects of hypoxia on the proliferation of adipose-derived stem cells and their differentiation into endothelial cells and investigate the role of PI3K/Akt pathway in this process. METHODS: Passage 3 adipose-derived stem cells of Wistar rats were divided into three groups according to the different culture conditions: normoxia group, hypoxia group, and hypoxia+LY294002 group. Three groups of cells were cultured for 24 hours in an incubator containing 20% O2. Cells in the hypoxia group were cultured in an incubator containing 2% O2, and those in the normoxia group were still cultured in an incubator containing 2% O2. Cells in the hypoxia+LY294002 group were cultured in medium supplemented with 25 mmol/L PI3K/Akt pathway inhibitor LY294002 under the hypoxic culture conduction. After 24 hours of culture, p-Akt expression was detected by Western blot assay to indicate the activation of PI3K/Akt pathway. The proliferation of adipose-derived stem cells was measured by CCK-8 method. Three groups of adipose-derived stem cells were induced to differentiate into vascular endothelial cells for 10 days. The differentiation of adipose-derived stem cells into endothelial cells was detected by qRT-PCR and anti-CD31 immunofluorescence staining. RESULTS AND CONCLUSION: Adipose-derived stem cells at passage 3 exhibited elongated fibroblast-like morphology. Cytometry analysis showed most of adipose-derived stem cells at passage 3 were negative for CD 34 and CD45 and positive for CD105 and could be induced towards osteogenic and adipogenic differentiation. The expression of p-Akt was significantly increased after hypoxic culture, which was inhibited obviously after adding LY294002. CCK-8 showed that the proliferation of adipose-derived stem cells in the hypoxia group was significantly higher than that in the normoxia group and hypoxia+LY294002 group (P < 0.05). The proliferation of adipose-derived stem cells in the hypoxia+LY294002 group was significantly lower than that in the hypoxia group (P < 0.05). The results of qRT-PCR and anti-CD31 immunofluorescence staining showed that the expression of endothelial cell gene and specific protein CD31 in the hypoxia group was significantly higher than that in the normoxia and hypoxia+LY294002 groups (P < 0.05). The expression of CD31 in the hypoxia+LY294002 group was significantly lower than that in the hypoxia group (P < 0.05), but it was significantly higher than that in the normoxia group (P < 0.05). These results suggest that the PI3K/Akt pathway plays an important role in hypoxia-induced cell proliferation and vascular endothelial cell differentiation of adipose-derived stem cells.