猪雌激素受体关联受体a基因的克隆、表达及其对脂肪细胞脂质聚集的影响

Cloning, expression of the porcine Estrogen-related receptor a gene and its effect on lipid accumulation in mature adipocytes

摘要:

雌激素受体关联受体a(Estrogen-related receptor a, ERRa)是调控机体能量代谢的关键转录调控因子, 其在白色脂肪组织中的作用尚不清楚。本研究旨在通过touch down-PCR方法克隆猪ERRα基因的ORF序列; 通过Western blotting和细胞免疫荧光染色法分析其在猪各组织及成熟脂肪细胞中的表达模式; 利用ERRa特异性抑制剂XCT790处理原代培养的猪成熟脂肪细胞, 探讨其对成熟脂肪细胞甘油三酯聚集的影响。结果显示, 所克隆的猪ERRα基因ORF序列长1269 bp (GenBank Accession No. FJ446485, 尚未公布), 编码422个氨基酸, 其核苷酸和氨基酸序列与其他物种高度同源; ERRa蛋白高表达于猪白色脂肪组织(White adipose tissue, WAT)、肾脏以及心脏中, 在脾脏中表达量较低; 细胞免疫荧光化学染色显示, ERRα蛋白广泛分布于脂肪细胞的细胞核和胞浆中; XCT790在10 mmol/L浓度时显著抑制了ERRa蛋白的表达和成熟脂肪细胞中甘油三酯的聚集。本研究将为有效调控体脂沉积提供新的靶点和理论参考。

Abstract:

Estrogen-related receptor a (ERRa) is an orphan nuclear receptor and functions as a key regulator of energy metabolism in high energy demand tissues. However, its role in white adipose tissue is largely unknown. In this study, we aim to clone the ORF sequence of pig ERRα with touch down-PCR, analyze the expression pattern of ERRa protein and its subcellular localization with Western blotting and cell immunofluorescence method respectively, and identify the effect of ERRα on lipid accumulation in mature porcine adipocytes with its specific inhibitor XCT790. The results showed that the ERRα ORF sequence is 1269 bp (GenBank Accession No. FJ446485, not published), and encode 422 amino acids. The homologies of ERRα nucleotide and amino acids sequences are high in different species. ERRα protein is highly expressed in pig white adipose tissue, kidney and heart, while remarkably lower in spleen. Cell immunofluorescence results indicated that ERRα protein distribute widely in adipocytes nucleus and cytoplasm. XCT790 significantly inhibited the expression of ERRα and lipid accumulation in porcine mature adipocyte. This study will provide new target and theoretical reference for fat deposition control.