Study on the expression patten of clock genes during adipogenic differentiation of porcine dedifferentiated fat cells
In order to investigate the expression pattern of clock gene during adipogenic differentiation,porcine dedifferentiated fat(DFAT)cells were subjected to adipogenic induction in vitro.The differentiation status of cells was observed under an inverted microscope on day 0,7 and 15 of induction respectively.Oil red O staining was used to observe lipid droplet accumulation.Real-time quantitative PCR was used to detect the relative mRNA expressions of clock genes(Bmal1,clock,Per2,Cry1 and Rev-erbα),key lipogenic factors(PPARγ),and adipocytokines(leptin and adiponectin)on day 0,7 and 15.ELISA was employed to measure the contents of leptin and adiponectin in the cell culture supernatant and analyze their secretion changes during adipogenic differentiation.The results showed that porcine DFAT cells before adipogenic induction exhibited spindle-shaped with uniform cytoplasm and no lipid droplet.By day 7 of induction,the cells gradually became oval,with small lipid droplets appearing in the cytoplasm.By day 15,the cells became more rounded,and the lipid droplets increased significantly and coalesced into more prominent structures.Oil red O staining showed red-stained lipid droplets within the cells.With the induction time,the relative mRNA expression levels of Bmal1 and Cry1 showed a gradual increase trend.Clock and Rev-erbα mRNA levels initially increased and then decreased,whereas Per2 expression decreased first and then increased.The relative mRNA expression levels of PPARγ,leptin and adiponectin also gradually increased.Porcine DFAT cells secreted leptin and adiponectin in vitro,with secretion levels increasing over the induction time.These results indicated that porcine DFAT cells had strong adipogenic differentiation ability in vitro.During the induction of lipogenesis,the expression patterns of mammalian Clock genes(Bmal1,Clock,Cry1,Per2 and Rev-erbα)exhibited different trends.Notably,Bmal1 and Cry1 showed expression trends similar to PPARγ,leptin and adiponectin,implying that PPARγ might play an important factor in linking the biological clock with adipogenesis and metabolism.
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