Abstract: Objective: To explore the effect of p62 gene deletion on the autophagy activity of human adipose-derived stromal cells (hADSCs), and to clarify the molecular mechanism of fat differentiation. Methods: The hADSCs were isolated and cultured from human thigh subcutaneous fat tissue by typeⅠcollagenase digestion method. Adipogenic induction of hADSCs was performed with 80 μmol·L^-1 oleic acid(OA) combined with dexamethasone and insulin. The hADSCs were divided into control group and p62 gene deletion group. The hADSCs in p62 gene deletion group were transfected with p62 shRNA lentiviral vectors. The hADSCs in control group were transfected with empty vectors. CCK-8 assay was used to draw the growth curves of cells in two groups,and the changes of cell proliferation activities were observed; Nile red staining was used to observe the lipid droplet formation(number of Nile red staining positive cells); Western blotting method was used to detect the expression levels of adipogenic marker transcription factor C/EBPα and microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ/Ⅰ (LC3Ⅱ/LC3Ⅰ).The autophagy activities (number of MDC staining positive cells) in two groups were detected by MDC staining. The mitochondrial autophagy activities of the cells in two groups were detected by fluorescence colocalization of LC3 and Mitotracker Red. Cyclosporin A(CsA) was used to inhibit the mitochondrial autophagy activities of the cells in two groups to induce adipogenesis, and the number of cells containing lipid droplets was counted under light microscope. Results: The isolated and cultured cells were polygonal or spindle-shaped, and the cells were arranged in a vortex after passage. A large number of lipid droplets were formed in the cells after OA-induced adipogenesis of hADSCs. The CCK-8 assay results showed that the number of cell proliferation activity in p62 gene deletion group 6 d after culture was significantly decreased (P<0.01).After adipogenesis induction,compared with control group, the number of Nile red staining positive cells in p62 gene deletion group was increased, the expression level of C/EBPα protein was increased (P<0.01); the number of MDC staining positive cells was significantly increased (P<0.05), and the expression levels of LC3Ⅱ/LCⅠproteins were increased (P<0.01); the pitting and granulation of LC3 in the cells was increased, and the colocalization with mitochondria was increased. The number of cells containing lipid droplets in p62 gene deletion group was reduced to a level close to that in control group under light microscope after CsA was used to inhibit the mitochondrial autophagy. Conclusion: The deletion of p62 gene can promote the adipogenesis of hADSCs by enhancing the total autophagy and mitochondrial autophagy.

Key words: adipose-derived stromal cells, adipogenesis, oleic acid, autophagy