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