摘要目的 探究棕色脂肪细胞分化过程中关键基因以及参照基因随分化进程的表达变化趋势.方法 根据随机数字表法随机选取20只4周龄雄性C57BL/6J小鼠,提取肩胛间区棕色脂肪组织.以高糖DMEM培养原代棕色前体脂肪细胞,以“鸡尾酒法”诱导培养基(含0.5 mmol/L异丁基甲基黄嘌呤、5 mmol/L地塞米松、320 nmol/L胰岛素、1 nmol/L三碘甲状腺原氨酸和0.125 mmol/L吲哚美辛)诱导分化.Trizol裂解法提取分化过程中(0、2、4、6d)棕色脂肪细胞总RNA,琼脂糖凝胶电泳验证总RNA质量.油红O染色,检测棕色脂肪细胞成熟情况,免疫荧光鉴定棕色脂肪细胞成熟标志物UCP1表达水平.实时荧光定量聚合酶链反应(RT-qPCR)法比较在棕色脂肪细胞中参照基因(GAPDH、β-actin、18S和PPIA)表达水平的恒定性,并检测棕色脂肪分化关键基因(UCPI、PRDM16、PGC-1α、EBF2、PPARγ和Cidea)的表达水平.结果 1.诱导第6天可见棕色脂肪细胞内密布细小脂滴,UCP1表达水平较高.2.琼脂糖凝胶电泳检测RNA质量,可见3条明亮带,提示RNA质量合格.3.在小鼠棕色脂肪分化过程中PPIA的表达水平相对恒定,可作为小鼠棕色脂肪分化合适的内参.4.棕色脂肪能量代谢相关基因UCP1、PRDM16、PGC-1α、EBF2、PPARγ和Cidea随着分化天数(0、2、4、6d)的增加表达量显著增加,差异有统计学意义(P均＜0.05).结论 原代棕色脂肪细胞分化过程中,内参基因PPIA表达恒定,是合适的内参基因.原代棕色分化关键基因UCP1、PRDM16、PGC-1α、EBF2、PPARγ和Cidea可作为合适的棕色脂肪细胞成熟标志基因.

abstractsObjective 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-1 α,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 andβ-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 differentiation key genes,such as UCP1,PRDM16,PGC-1α,EBF2,PPARγ and cidea,Could be used as suitable mature marker genes during adipogenesis.