过表达NR4A1促进山羊皮下脂肪细胞分化

Overexpression of NR 4A1 Promotes Goat Subcutaneous Adipocytes Differentiation

旨在克隆山羊NR4A1基因的CDS区序列,明确其组织和细胞表达模式,以及探究过表达NR4A1基因对山羊皮下脂肪细胞分化的影响。本试验利用双酶切法构建山羊过表达载体pcDNA3.1-NR4A1。以1周岁简州大耳羊(n=5)为试验动物。利用RT-PCR方法和实时荧光定量PCR(real-time quantitative PCR,qPCR)技术克隆NR4A1基因编码区序列并明确其时空表达特性,再将山羊pcDNA3.1-NR4A1载体转染皮下脂肪细胞使NR4A1过表达,利用形态学方法检测过表达后脂滴聚集的变化,同时采用qPCR方法检测脂肪分化标志基因相对表达水平的变化。结果获得山羊NR4A1基因的编码区序列是1797 bp,编码598个氨基酸;NR4A1在山羊各组织中广泛表达,且在山羊背最长肌中的相对表达水平最高(P<0.01),在山羊皮下脂肪细胞分化60 h表达量最高(P<0.01);过表达NR4A1基因显著促进山羊皮下脂肪细胞的脂滴积累,并且显著提高C/EBPα、C/EBPβ、PPARγ、LPL、SREBP1和AP2的相对表达水平(P<0.05)。NR4A1基因可能是山羊皮下脂肪细胞分化的正调控因子,且可能是协同脂肪分化标志基因的表达量来实现的。

The aim of this research was to clone the goat NR 4A1 gene CDS sequence,clarify its tissue and cell expression patterns,and explore the effect of overexpression of NR 4A1 gene on goat subcutaneous adipocyte differentiation.In this experiment,the goat overexpression vector pcDNA3.1-NR4A1 was constructed by double digestion.Five healthy Jianzhou big ear goats of one-year-old were used as experimental animals.The coding region sequence of NR 4A1 gene was cloned through RT-PCR.Relative expression level of NR 4A1 gene in different tissues and subcutaneous adipocytes at different stages were detected by real-time quantitative PCR(qPCR).By transfecting pcDNA3.1-NR4A1 vector into subcutaneous adipocytes to overexpress NR 4A1,and changes of lipid droplet aggregation after overexpression was detected by morphology,at the same time,the relative expression level of adipose differentiation marker genes was detected by qPCR.The results showed that the coding region sequence of goat NR 4A1 gene was 1797 bp,encoding 598 amino acids.NR 4A1 widely expressed in different tissues of goats and had the highest expression level in the longissimus dorsi(P<0.01),and NR 4A1 expression level was the highest in goat subcutaneous adipocytes at 60 h of differentiation(P<0.01).The overexpression of NR 4 A 1 significantly promoted the accumulation of lipid droplets in subcutaneous adipocytes of goats,and significantly upregulated the relative expression level of C/EBPα,C/EBPβ,PPARγ,LPL,SREBP 1 and AP 2(P<0.05).NR 4A1 maybe a positive regulator for goat subcutaneous adipocytes differentiation by regulating the expression of adipocyte differentiation marker genes.