慢病毒介导shRNA干扰颗粒体蛋白前体(GRN)促进猪前体脂肪细胞分化

为研究颗粒体蛋白前体(granulin,GRN)在脂肪细胞发育过程中的作用,本实验构建了GRN短发夹RNA(short hairpin RNA,shRNA)慢病毒干扰载体,包装并感染猪前体脂肪细胞,采用油红O染色、油红O提取比色法检测猪前体脂肪细胞分化情况,采用Real-time PCR法检测成脂关键基因mRNA的表达变化情况。结果显示,GRN慢病毒干扰载体病毒滴度在5×107TU/mL以上,病毒感染前体脂肪细胞后显著降低了GRN的表达,shRNA2干扰效率最高,达到76%,沉默GRN后能够促进猪前体脂肪细胞分化;脂肪细胞分化标志基因过氧化物酶体增殖物激活受体γ(PPARγ)、固醇调节原件结合蛋白(SREBP-1c)、脂肪细胞型脂肪酸结合蛋白(ap2)和脂蛋白酯酶(LPL)基因mRNA表达量均升高。结果表明,降低GRN基因表达促进了猪前体脂肪细胞分化,揭示GRN在猪前体脂肪细胞分化过程中可能起到抑制作用。

Lentivirus Mediated Granulin shRNA Interference Silencing Promotes Pig Preadipocyte Differentiation

In order to investigate the role of granulin(GRN) during the development of adipocyte,we constructed lentivirus interfering vector targeting on porcine GRN,packaged and infected the pig preadipocyte,differentiation state of preadipocyte was detected by Oil Red O staining and Oil Red O extraction assay,the expression levels of adipocyte differentiation marker genes were analysed by Real-time PCR.The results showed that the virus titer of the lentivirus reached 5×107 TU/mL,and the GRN expression level was remarkably decreased after lentivirus infection,of which,GRN mRNA expression was suppressed by approximate 76% by short hairpin RNA(shRNA2),moreover,adipocyte differentiation was promoted,and adipocyte differentiation marker genes,such as peroxisome proliferator activated receptor γ(PPARγ),sterol regulatory element binding protein 1(SREBP-1C),adipocyte-type fatty acid-binding protein(ap2) and lipoprotein lipase(LPL) mRNA expression levels were all increased.Taken together these data suggested that knocking down of GRN promoted adipocyte differentiation,and GRN may play a negative role in pig preadipcoyte differentiation.