目的探讨硫酸脱氢表雄酮(DHEA-S)对3T3-L1前脂肪细胞株分化的影响及可能的分子机制。方法以3T3-L1细胞株为研究对象,先观察不同时间点不同剂量的DHEA-S对3T3-L1细胞活力的影响,确定合适的干预浓度。3T3-L1细胞分为DMSO组(对照组)和DHEA-...目的探讨硫酸脱氢表雄酮(DHEA-S)对3T3-L1前脂肪细胞株分化的影响及可能的分子机制。方法以3T3-L1细胞株为研究对象,先观察不同时间点不同剂量的DHEA-S对3T3-L1细胞活力的影响,确定合适的干预浓度。3T3-L1细胞分为DMSO组(对照组)和DHEA-S组,观察DHEA-S对3T3-L1前脂肪细胞分化的影响。通过测定甘油醛3-磷酸脱氢酶(G3PDH)活性和用油红O染色计算的脂肪细胞分化率,评估脂肪细胞分化的变化;用real time-PCR法观察诱导前(第0天)和诱导后不同时间点(第1,2,4,6,10,14天)目的基因过氧化物酶体增殖物激活受体-γ(PPARγ)、固醇调节元件结合蛋白-1c(SREBP1-c)、CCAAT增强子结合蛋白家族(C/EBPs)、11β-羟类固醇脱氢酶1(11HSDB1)、激素敏感性脂肪酶(HSL)和脂蛋白酯酶(LPL)mRNA表达的变化。结果MTT实验结果显示DHEA-S在50μmol/L时对细胞活力影响<30%,对后续的干预影响较小。分化后第4天对照组和DHEA-S组的G3PDH活性分别为(380.83±28.43)nmol/(min·mg)和(253.07±27.67)nmol/(min·mg),分化后第10天对照组和DHEA-S组的G3PDH活性为(573.93±17.5)nmol/(min·mg)和(398.17±24.05)nmol/(min·mg),差异均具有统计学意义(P<0.01)。油红O染色计算脂肪细胞分化率对照组和DHEA-S组在诱导后第4天分别为39.09%±5.51%和15.66%±0.75%,第10天分别为78.82%±3.37%和49.09%±9.75%,差异也有统计学意义(P<0.01)。real time PCR检测结果显示,DHEA-S显著抑制脂肪细胞分化相关的关键基因(PPARγ、ADD1/SREBP1-c、C/EBPs、11HSDB1、LPL和HSL)mRNA的表达。结论DHEA-S(50μmol/L)在体外可能通过抑制11HSDB1的表达,减少局部活性皮质醇,进而下调其他脂肪细胞分化的关键基因,抑制3T3-L1细胞的分化。展开更多
CCAAT/enhancer binding protein α （C/EBPα） is a transcriptional regulatory factor that inhibits cell proliferation, and alternative translational initiation produces two polypeptides, C/EBPαp30 and C/EBPαp42. By ...CCAAT/enhancer binding protein α （C/EBPα） is a transcriptional regulatory factor that inhibits cell proliferation, and alternative translational initiation produces two polypeptides, C/EBPαp30 and C/EBPαp42. By expression profiling, it was revealed that C/EBPap30 specifically inhibited a unique set of genes, including MPP 11, p84N5 and SMYD2, which were not affected by C/EBPαp42 in both QSG-7701 hepatocyte cell line and QGY-7703 hepatoma cells. Semiquantitative RT-PCR analysis independently confirmed these results. Chromatin immunoprecipitation assay showed that C/EBPαp30 bound to the promoters of these genes more strongly than C/EBPαp42. In clinical hepatoma samples in which C/EBPα was downregulated, all three genes specifically inhibited by C/EBPαp30 were upregulated. However, repression of MPP 11, p84N5 and SMYD2 genes might not be directly involved in C/EBPαp30-mediated growth inhibition. Our data suggest that C/EBPαp30 regulates a unique set of target genes and is more than a dominant-negative regulator of C/EBPαp42.展开更多
文摘目的探讨硫酸脱氢表雄酮(DHEA-S)对3T3-L1前脂肪细胞株分化的影响及可能的分子机制。方法以3T3-L1细胞株为研究对象,先观察不同时间点不同剂量的DHEA-S对3T3-L1细胞活力的影响,确定合适的干预浓度。3T3-L1细胞分为DMSO组(对照组)和DHEA-S组,观察DHEA-S对3T3-L1前脂肪细胞分化的影响。通过测定甘油醛3-磷酸脱氢酶(G3PDH)活性和用油红O染色计算的脂肪细胞分化率,评估脂肪细胞分化的变化;用real time-PCR法观察诱导前(第0天)和诱导后不同时间点(第1,2,4,6,10,14天)目的基因过氧化物酶体增殖物激活受体-γ(PPARγ)、固醇调节元件结合蛋白-1c(SREBP1-c)、CCAAT增强子结合蛋白家族(C/EBPs)、11β-羟类固醇脱氢酶1(11HSDB1)、激素敏感性脂肪酶(HSL)和脂蛋白酯酶(LPL)mRNA表达的变化。结果MTT实验结果显示DHEA-S在50μmol/L时对细胞活力影响<30%,对后续的干预影响较小。分化后第4天对照组和DHEA-S组的G3PDH活性分别为(380.83±28.43)nmol/(min·mg)和(253.07±27.67)nmol/(min·mg),分化后第10天对照组和DHEA-S组的G3PDH活性为(573.93±17.5)nmol/(min·mg)和(398.17±24.05)nmol/(min·mg),差异均具有统计学意义(P<0.01)。油红O染色计算脂肪细胞分化率对照组和DHEA-S组在诱导后第4天分别为39.09%±5.51%和15.66%±0.75%,第10天分别为78.82%±3.37%和49.09%±9.75%,差异也有统计学意义(P<0.01)。real time PCR检测结果显示,DHEA-S显著抑制脂肪细胞分化相关的关键基因(PPARγ、ADD1/SREBP1-c、C/EBPs、11HSDB1、LPL和HSL)mRNA的表达。结论DHEA-S(50μmol/L)在体外可能通过抑制11HSDB1的表达,减少局部活性皮质醇,进而下调其他脂肪细胞分化的关键基因,抑制3T3-L1细胞的分化。
文摘CCAAT/enhancer binding protein α （C/EBPα） is a transcriptional regulatory factor that inhibits cell proliferation, and alternative translational initiation produces two polypeptides, C/EBPαp30 and C/EBPαp42. By expression profiling, it was revealed that C/EBPap30 specifically inhibited a unique set of genes, including MPP 11, p84N5 and SMYD2, which were not affected by C/EBPαp42 in both QSG-7701 hepatocyte cell line and QGY-7703 hepatoma cells. Semiquantitative RT-PCR analysis independently confirmed these results. Chromatin immunoprecipitation assay showed that C/EBPαp30 bound to the promoters of these genes more strongly than C/EBPαp42. In clinical hepatoma samples in which C/EBPα was downregulated, all three genes specifically inhibited by C/EBPαp30 were upregulated. However, repression of MPP 11, p84N5 and SMYD2 genes might not be directly involved in C/EBPαp30-mediated growth inhibition. Our data suggest that C/EBPαp30 regulates a unique set of target genes and is more than a dominant-negative regulator of C/EBPαp42.
