摘要
【目的】探究鸭原代肝细胞脂质含量与碳水化合物反应元件结合蛋白基因(ChREBP)表达的关联性,为深入研究ChREBP基因对畜禽脂质代谢的遗传调控机制提供参考依据。【方法】采用Ⅳ型胶原酶消化法分离孵化21 d的鸭胚原代肝细胞,以DMEM高糖培养基进行培养。利用RT-PCR扩增鸭ChREBP基因编码区(CDS)序列,构建携带His标签的真核表达载体pEGFP-N3-ChREBP-His,通过脂质体法分别转染鸭胚原代肝细胞和人前列腺癌细胞(PC3);采用His标签检测试剂盒检测ChREBP基因的表达量,以脂质指标测定试剂盒测定甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白(HDL)和低密度脂蛋白(LDL)的含量,并分析鸭胚原代肝细胞和PC3细胞中ChREBP基因表达与脂质指标含量的相关性。【结果】以构建的真核表达载体pEGFP-N3-ChREBP-His转染鸭原代肝细胞和PC3细胞均能发出绿色荧光,表明携带His标签的ChREBP基因能在鸭原代肝细胞和PC3细胞中表达。ChREBP基因表达量与鸭原代肝细胞和PC3细胞中的TG、TC和HDL含量均呈极显著正相关(P<0.01,下同),与LDL含量呈极显著负相关。【结论】以构建的真核表达载体pEGFP-N3-ChREBP-His转染鸭原代肝细胞和PC3细胞后均能发出绿色荧光,说明ChREBP基因表达对鸭原代肝细胞和PC3细胞中的脂质代谢作用机理相似,可促进细胞脂质累积。
【Objective】To explore the relationship between lipid content and carbohydrate response element binding protein(ChREBP)gene expression in primary duck hepatocytes,so as to provide reference for further study of the genetic regulation mechanism of ChREBP gene on lipid metabolism in livestock and poultry.【Method】Primary hepatocytes of 21-day-old duck embryos were isolated by type IV collagenase digestion and cultured in DMEM high glucose medium.The sequence of duck ChREBP gene coding region(CDS)was amplified by RT-PCR,and the eukaryotic expression vector pEGFP-N3-ChREBP-His with His tag was constructed and transfected into duck embryo primary hepatocytes and human prostate cancer cells(PC3)by liposome method.The expression of ChREBP gene was detected by His label detection kit,and the contents of triglyceride(TG),total cholesterol(TC),low density lipoprotein(LDL)and high density lipoprotein(HDL)were measured by lipid index assay kit.The correlation between ChREBP gene expression and lipid index content in primary duck embryo hepatocytes and PC3 cells was analyzed.【Result】The constructed eukaryotic expression vector pEGFP-N3-ChREBP-His could emit green fluorescence in duck primary hepatocytes and PC3 cells,indicating that ChREBP gene with His tag could be expressed in duck primary hepatocytes and PC3 cells.The expression of ChREBP gene was very significantly positively correlated with the contents of TG,TC and HDL in primary duck hepatocytes and PC3 cells(P<0.01,the same below),and very significantly negatively correlated with the content of LDL.【Conclusion】The constructed eukaryotic expression vector pEGFP-N3-ChREBP-His can emit green fluorescence after transfection into duck primary hepatocytes and PC3 cells,indicating that the mechanism of ChREBP gene expression on lipid metabolism in duck primary hepatocytes and PC3 cells is similar,which can promote cellular lipid accumulation.
作者
谭光辉
覃媛钰
王单单
李万贵
罗华伦
吴磊
李杰章
张依裕
TAN Guang-hui;QIN Yuan-yu;WANG Dan-dan;LIWan-gui;LUO Hua-lun;WU Lei;LI Jie-zhang;ZHANG Yi-yu(College of Animal Science,Guizhou University/Key Laboratory of Animal Genetics,Breeding and Reproduction in the Plateau Mountainous Region,Ministry of Education,Guiyang 550025,China;The GrassWorkstation of Liupanshui City,Liupanshui,Guizhou 553001,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2020年第4期937-944,共8页
Journal of Southern Agriculture
基金
国家自然科学基金项目(31760663)
贵州省生物学一流学科建设项目(GNYL〔2017〕009)
贵州省优秀青年科技人才补助项目(黔科合平台人才〔2017〕5788)。
