RNA干扰DGAT1和DGAT2在猪肝细胞和肌内前体脂肪细胞中表达的研究

Study on RNA Interference Expression of DGAT1 and DGAT2 in Porcine Hepatocytes and Intramuscular Precursor Adipocytes

本研究旨在探索DGAT1和DGAT2基因表达对猪脂肪沉积效率的影响。以3日龄宗地花猪为实验动物,针对DGAT1和DGAT2基因m RNA序列设计6对sh RNA序列,构建RNA干扰表达载体,将DGAT1和DGAT2的sh RNA干扰载体分别转染至猪的肝细胞和肌内前体脂肪细胞中,采用RT-q PCR筛选出肝细胞和肌内前体脂肪细胞中的最佳sh RNA干扰载体,将最佳干扰载体再次转染肝细胞和肌内前体脂肪细胞,测定细胞代谢液中甘油三酯和总胆固醇含量变化。结果表明:所构建的干扰载体对肝细胞中DGAT1和DGAT2基因的最高抑制效率分别为78.45%和87.52%,对肌内前体脂肪细胞中DGAT1和DGAT2基因的最高抑制效率分别为82.24%和89.82%;肝细胞和肌内前体脂肪细胞培养液中甘油三酯和总胆固醇的含量均降低,其中转染p GPU6/GFP/Neo-DGAT2-1实验组的肝细胞培养液中甘油三酯含量显著低于空白组(P<0.05)。结果提示,抑制DGAT1和DGAT2基因的表达,可降低猪肝细胞和肌内前体脂肪细胞代谢液中甘油三酯和总胆固醇的含量。

The purpose of this study was to explore the effect of DGAT1 and DGAT2 genes expression on fat deposition efficiency in pigs.In this study,6 pairs of shRNA sequences were designed for mRNA sequences of DGAT1 and DGAT2 genes,and RNA interference expression vectors were constructed by used 3 days old Zongdihua pigs as experimental animals,the shRNA interference vectors of DGAT1 and DGAT2 were transfected into porcine hepatocytes and intramuscular precursor adipocytes respectively,the best shRNA interference vectors in hepatocytes and intramuscular precursor adipocytes were selected by RT-qPCR retransfection of optimal interference vectors into Hepatocytes and Intramuscular Precursor Adipocytes,triglyceride and total cholesterol contents changes were tested in cells metabolic fluid.The results indicated that the maximum inhibitory efficiency of the constructed interference vectors on DGAT1 and DGAT2 genes in Hepatocytes was 78.45%and 87.52%,respectively,and that on DGAT1 and DGAT2 genes in intramuscular precursor adipocytes was 82.24%and 89.82%,respectively,the contents of triglyceride and total cholesterol in Hepatocytes and Intramuscular precursor adipocytes were decreased in metabolic fluid.The contents of triglyceride in hepatocytes metabolic fluid transfected with pGPU6/GFP/Neo-DGAT2-1 group was significantly lower than that of blank group(P<0.05).Inhibiting the expression of DGAT1 and DGAT2 genes could decrease the contents of triglyceride and total cholesterol in the metabolites of porcine Hepatocytes and Intramuscular Precursor Adipocytes.