敲减MC4R表达对牛胎儿成纤维细胞CMS系统关键因子的影响  

Effect of the RNAi-based MC4R Silencing on the Expression of Key Factors in Central Melanocortin System in Bovine Fibroblasts

作　　者：黄萌[1,2] 许尚忠[1,2] 李俊雅[2] 高雪[2] 陈金宝[2] 

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100 [2]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《中国生物化学与分子生物学报》2011年第3期267-272,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家转基因新品种重大专项(No.2008ZX08007-002;No.2009ZX08009-157B)~~

摘　　要：为获得敲减黑素皮质素4受体(melanocortin-4 receptor,MC4R)基因的牛胎儿成纤维细胞,并探讨其在能量平衡神经调节系统中的作用,将构建成功并已鉴定为有效序列的短发夹状RNA(short hairpin RNA,shRNA)真核表达载体pGSH1-GFP-MC4R,利用阳离子脂质体转染牛胎儿成纤维细胞并使用G418筛选稳定转染细胞株.利用实时荧光定量和Western印迹检测MC4R及中枢黑素皮质素系统(central melanocortin system,CMS)关键因子的表达水平变化.结果表明,在稳定转染的牛胎儿成纤维细胞系中,MC4R表达显著抑制,瘦蛋白(leptin)和阿黑色素原(POMC)表达下调,黑素皮质素拮抗物agouti相关蛋白(AGRP)和MC3R表达上调,而神经肽Y(NPY)表达无明显改变.综上所述,本研究成功获得了敲减MC4R基因表达的牛胎儿成纤维细胞.相关基因表达水平检测结果提示,MC4R的表达水平对CMS系统中的各关键基因的表达有不同的抑制或促进影响.To explore the effect of MC4R in the energy homeostasis neuroregulation system,the melanocortin-4 receptor（MC4R） knockdown bovine fetal fibroblast cells were prepared.The short hairpin RNA（shRNA） eukaryotic expression vectors for MC4R was constructed and transfected into bovine fetal fibroblasts using lipofectamineTM 2000.After screened with geneticin（G418）,transfected stable cell clones were obtained.The expression of MC4R and other key factors of the central melanocortin system（CMS） were evaluated by SYBR green Ⅰ real-time RT-PCR and Western blotting.In stablely transfected MC4R knockdown bovine fetal fibroblasts,MC4R expression was significantly reduced and the Leptin and proopiomelanocortin（POMC） expression were also down-regulated,but the agouti-related protein（AGRP） and MC3R were up-regulated.No significant changed of the neuropeptide Y（NPY） expression was observed.In conclusion,this study implied that MC4R was connected with the inhibition or enhancement of the key genes in the CMS.

关 键 词：黑素皮质素4受体(MC4R) RNA干扰 牛胎儿成纤维细胞 中枢黑素皮质素系统(CMS) 能量平衡 

分 类 号：Q78[生物学—分子生物学]