慢病毒介导shRNA干扰RBP4促进猪前体脂肪细胞成脂分化

摘要

To investigate the role of RBP4 during the differentiation of porcine preadipocytes and the biological function of RBP4,we constructed porcine RBP4 lentivirus interfering vector,then infected the porcine preadipocytes.The changes in morphology and gene expression levels were studied to evaluate the effects of RBP4 expression on preadipocyte differentiation by BODIPY,Oil Red O staining and real-time PCR analysis.The results showed that RBP4 lentivirus interfering vector was constructed correctly,virus titer was 6.5 × 107 pfu · mL-1.RBP4 mRNA and protein expression was remarkably reduced by approximate 60％ in porcine preadipocytes infected with shRBP4.Moreover,preadipocytes differentiation was promoted and the mRNA expression levels of PPARγ,SREBP-lc and aP2 were increased in adipocytes by knocking down of RBP4.Therefore,adipogenic differentiation of porcine preadipocytes are inhibited by RBP4,which would help the further exploration of the biological mechanism of RBP4 during the adipogenesis.%为研究视黄醇结合蛋白4(retinol binding protein 4,RBP4)对猪前体脂肪细胞分化的影响,笔者构建了RBP4重组慢病毒干扰载体,包装并感染猪前体脂肪细胞,采用BODIPY和油红O染色以及real-time PCR等方法,从形态学和基因表达水平检测抑制RBP4表达对成脂分化的作用.研究结果显示,RBP4重组慢病毒干扰载体构建成功,病毒滴度达到6.5×107pfu·mL-1,能显著抑制猪前体脂肪细胞中RBP4的表达水平(干扰效率在60％以上).同时,干扰RBP4能促进猪前体脂肪细胞分化,增加成脂关键基因PPARγ、SREBP-1c和aP2的mRNA表达.本研究结果表明,RBP4对猪前体脂肪细胞分化有抑制作用,为进一步研究RBP4对猪前体脂肪细胞分化的作用机制奠定基础.