奶牛乳腺上皮细胞中SCAP和SREBP1蛋白调控SCD基因表达的作用研究

摘要

旨在研究奶牛乳腺上皮细胞中固醇调节元件结合蛋白裂解激活蛋白(SCAP)对固醇调节元件结合蛋白(SREBP1)调控的硬脂酰辅酶A去饱和酶基因(SCD)表达的影响.培养奶牛乳腺上皮细胞,在细胞中转染奶牛SCD基因启动子载体,同时转染SREBP1和SCAP真核表达载体作为处理,采用双荧光素酶报告基因系统检测转染SREBP1和SCAP对SCD基因启动子活性的影响;采用免疫荧光技术观察SREBP1在细胞核的表达,采用荧光定量PCR检测SCD基因mRNA的表达.结果表明,与转染pcDNA3.1载体的对照组相比,转染SCAP对SCD启动子活性无显著影响;转染SREBP1和共转染SCAP/SREBP1极显著增加SCD基因启动子活性值(P＜0.01),并且SCAP的转染剂量与SCD的启动子活性值之间具有极显著的量效关系(P＜0.01);在乳腺上皮细胞中转染SCAP后,能够增强SREBP1在细胞核的表达;细胞转染SREBP1和共转染SCAP/SREBP1后,SCD基因mRNA的表达分别显著上调1.23倍和1.54倍(P＜0.05).本研究表明,奶牛SCAP可以增加SREBP1蛋白在细胞核中的表达,促进对SCD基因的转录激活作用.%The aim of this study was to investigate the effect of sterol regulatory element-binding protein cleavage activating protein (SCAP) on the expression of stearoyl-CoA desaturase(SCD) gene regulated by steroid regulatory element binding protein (SREBP1) in dairy mammary epithelial cells.The dairy mammary epithelial cells were cultured and transfected with SCD gene promoter vectors,then SREBP1 and SCAP eukaryotic expression vectors were transfected into cells as treatment factor.The SCD promoter activity was detected by dual luciferase reporter gene system.The expression of SREBP1 in nucleus was observed by immunofluorescence.qRT-PCR was used to measure the mRNA expression of SCD gene.The results showed that,compared with pcDNA3.1 control group,transfection of SCAP had no significant effect on SCD promoter activity.Transfection of SREBP1 and co-transfection of SCAP/SREBP1 significantly increased the SCD promoter activity (P＜0.01).There was a significant dose-response relationship between the transfection dose of SCAP and the promoter activity of SCD (P＜ 0.01).The expression of SREBP1 in nucleus could be enhanced by SCAP transfection in mammary epithelial cells.The mRNA expression of SCD was up-regulated by 1.23-fold and 1.54-fold (P ＜0.05) after transfected SREBP1 or co-transfected SCAP/SREBP1,respectively.In conclusion,SCAP can increase the expression of SREBP1 protein in nucleus which enhance the transcriptional activation of SCD gene.