Changes in expression levels of brown adipocyte differentiation associated with key genes and reference controls during adipogenesis

Objective To investigate the expression changes in the key differentiation-associated genes and reference genes during the progress of primary brown phagocyte adipocytes. Methods Twenty 4-week-old male C57BL/6J mice were selected randomly and brown adipose tissues were extracted from interscapular area.These brown preadipocytes were cultured in high glucose dulbecco′s modified eagle media, and induced in cocktail culture media (containing 0.5 mmol/L isobutylmethylxanthine, 5 mmol/L Dexamethasone, insulin 320 nmol/L, 1 nmol/L three iodine thyroid original amino acid, and 0.125 mmol/L Indomethacin). The total RNA of brown fat cells in the differentiation process(0, 2, 4, 6 day) were extracted by Trizol RNA isolation assay respectively.And then the RNA quality was verified by agarose gel electrophoresis.Oil red O staining was conducted to detect the maturation of brown fat cells.UCP1, the maker of mature brown fat cells, was detected by immunofluorescence assay.The expression of reference (GAPDH, β-actin, 18S and PPIA) and brown adipocyte differentiation associated genes(UCP1, PRDM16, PGC-1α, EBF2, PPARγ and Cidea) were detected by real-time quantitative PCR(RT-qPCR) method. Results (1)On day 6 of introduction, brown adipocytes were filled with small lipid droplets, and the protein expression of UCP1 increased significantly detected by immunofluorescence.(2)Agarose gel electrophoresis showed 3 bright bands, suggesting a fine RNA quality.(3)The relative gene expression levels of GAPDH, 18S and β-actin existed variations, wheras the expression level of PPIA maintained stable, indicating that PPIA could be used as a reference for differentiation of brown adipose tissues in mouse models during adipogenesis.(4)The expression of brown adipocyte differentiation-associated genes (UCP1, PRDM16, PGC-1α, EBF2, PPARγ and Cidea) increased gradually as time passed(0, 2, 4, 6 day) significantly(all P<0.05). Conclusions PPIA is a suitable reference gene for brown adipocyte differentiation.Brown differen-tiation key genes, such as UCP1, PRDM16, PGC-1α, EBF2, PPARγ and cidea, Could be used as suitable mature marker genes during adipogenesis. Key words: Brown adipocyte; Adipogenesis; Real-time quantitative PCR; Marker gene of brown adipocyte