鸭SCD1基因过表达对原代肝细胞脂质代谢的效应研究
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摘要
硬脂酰辅酶A去饱和酶1(Stearoy-Co A desaturase 1,SCD1)是肝细胞合成单不饱和脂肪酸的限速酶,对机体脂肪酸的组成具有重要调控作用。为了探究鸭(Anas platyrhynchos)SCD1基因对其脂肪沉积的遗传调控机制,本实验采用Ⅳ型胶原酶消化肝脏分离培养法分离21日鸭胚原代肝细胞并进行培养,构建携带HIS标签的p EGFP-N3-SCD1真核过表达载体,转染鸭胚原代肝细胞,利用HIS标签检测试剂盒测定SCD1基因在鸭胚原代肝细胞中的过表达量,探讨其过表达对鸭原代肝细胞脂质指标的效应。实验结果显示肝细胞初分离效果良好,细胞培养至96 h基本贴满培养板,p EGFP-N3-SCD1重组质粒在肝细胞中能稳定过表达并发出绿色荧光,SCD1基因的过表达与总胆固醇(total cholesterol,TC)及低密度脂蛋白(low density lipoprotein,LDL)含量呈极显著正相关(P<0.01),与高密度脂蛋白(high density lipoprotein,HDL)含量呈极显著负相关(P<0.01),与甘油三酯(triglyceride,TG)则不相关(P>0.05)。结果表明:Ⅳ型胶原酶消化肝脏提取法能成功分离培养鸭原代肝细胞,p EGFP-N3-SCD1重组质粒转染效果良好,SCD1基因过表达对鸭原代肝细胞的脂质代谢具有调控作用,这对今后进一步研究SCD1对肉鸭脂肪沉积的遗传调控机制具有重要的指导意义。
Stearoyl-Co A desaturase 1(SCD1) is a rate-limiting enzyme for the synthesis of monounsaturated fatty acids in hepatocytes, and plays an important regulation role in the composition of fatty acids in the body.In order to explore the genetic regulation mechanism of SCD1 gene on the fat deposition of poultry, 21 days embryo duck(Anas platyrhynchos) hepatocytes were extracted by the method of type Ⅳ collagenaseand. The p EGFP-N3-SCD1 eukaryotic over-expression vector was constructed and transferred into the hepatocytes. The over-expression of SCD1 gene in duck hepatocytes was measured by HIS labeling assay kit to understand the effect of SCD1 on lipid metabolism. The experimental results showed that initial separation of hepatocyteswas good, and the cells were basically covered with culture plates until 96 h, The recombinant plasmid p EGFP-N3-SCD1 and green fluorescence could over-expression stably in the primary hepatocytes of the ducks. The correlation between over-expression and lipid index showed that the expression of SCD1 was positively correlated with total cholesterol(TC) and low density lipoprotein(LDL)(P<0.01), The content of high density lipoprotein(HDL) was significantly negative correlation(P<0.01) but triglyceride(TG)(P>0.05). The results showed that the recombinant plasmid p EGFP-N3-SCD1 could be successfully isolated and purified from the liver extraction by using type Ⅳ collagenase. Over-expression of SCD1 gene had a regulatory function for lipid metabolism of duck primary hepatocytes and it is useful for the future study about the genetic regulation mechanism of SCD1 on duck fat deposition.
Stearoyl-Co A desaturase 1(SCD1) is a rate-limiting enzyme for the synthesis of monounsaturated fatty acids in hepatocytes, and plays an important regulation role in the composition of fatty acids in the body.In order to explore the genetic regulation mechanism of SCD1 gene on the fat deposition of poultry, 21 days embryo duck(Anas platyrhynchos) hepatocytes were extracted by the method of type Ⅳ collagenaseand. The p EGFP-N3-SCD1 eukaryotic over-expression vector was constructed and transferred into the hepatocytes. The over-expression of SCD1 gene in duck hepatocytes was measured by HIS labeling assay kit to understand the effect of SCD1 on lipid metabolism. The experimental results showed that initial separation of hepatocyteswas good, and the cells were basically covered with culture plates until 96 h, The recombinant plasmid p EGFP-N3-SCD1 and green fluorescence could over-expression stably in the primary hepatocytes of the ducks. The correlation between over-expression and lipid index showed that the expression of SCD1 was positively correlated with total cholesterol(TC) and low density lipoprotein(LDL)(P<0.01), The content of high density lipoprotein(HDL) was significantly negative correlation(P<0.01) but triglyceride(TG)(P>0.05). The results showed that the recombinant plasmid p EGFP-N3-SCD1 could be successfully isolated and purified from the liver extraction by using type Ⅳ collagenase. Over-expression of SCD1 gene had a regulatory function for lipid metabolism of duck primary hepatocytes and it is useful for the future study about the genetic regulation mechanism of SCD1 on duck fat deposition.
引文
蔡德丰,范建高,马东礼,等.2014.硬脂酰辅酶A去饱和酶-1过表达影响高脂诱导的肝细胞凋亡[J].中华肝脏病杂志,22(1):48-52.(Cai D F,Fan J G,Ma D L,et al2014.SCD1 over-expression inhibits palmitic acid-induced apoptosis of rat BRL hepatocytes[J].Chinese Journal of Hepatology,22(1):48-52.)
李万贵,张依裕,王单单,等.2016.鸭SCD1基因对PC3细胞脂质性状的效应分析[J].基因组学与应用生物学,35(9):2355-2360.(Li W G,Zhang Y Y,Wang D D,et al2016.Effect analysis of duck SCD1 gene on lipid traits of PC3 cells[J].Genomics and Annlied Aioloev,35(9)2355-2360.)
李卫华,杨佳欣.2014.硬脂酰辅酶A去饱和酶1与恶性肿瘤的研究进展[J].中国肿瘤临床,41(17):1131-1134(Li W H,Yang J X.2014.Development in the study of the correlation between stearoyl-coenzyme A desaturase1 and cancer progression[J].Chinese Journal of Clinica Oncology,41(17):1131-1134.)
刘三凤,任军,郭源梅,等.2008.鸡SCD和Sirt1基因的SNP搜寻及其遗传多样性分析[J].畜牧兽医学报,39(3):278-285.(Liu S F,Ren J,Guo Y M,et al.2008Identification of Single nucleotide polymorphisms of SCD and evaluation of their genetic diversities in chickens[J].Acta Veterinaria et Zootechnica Sinica,39(3)278-285.)
魏聪.2016.天府肉羊SCD1基因在肌肉中的表达与棕榈油酸和油酸含量的相关性研究[D].硕士学位论文,四川农业大学,导师:徐刚毅,pp.52-53.(Wei C.2016.Expression of the SCD1 gene and its relationship with the palmitoleic acid and oliec acid composition of tianfu goat[D].Thesis for M.S.,Sichuan Agricultural University,Suppervisor:Xu G Y,pp.52-53.)
徐雅玲,黄巨恩,梁菁,等.2016.改构型酸性成纤维细胞生长因子对紫杉醇诱导体外大鼠肝细胞损伤保护作用的体外实验研究[J].暨南大学学报(自然科学与医学版),37(1):44-48.((Xu Y L,Huang J E,Liang J,et al.2016.Protective effects of Ma FGF on the injury of hepatocytes induced by taxol in vitro[J].Journal of Jinan University(Natural Science and Medicine edition),37(1):44-48.)
余凯凡.2010.不同品种猪肌肉脂肪酸组成的比较及相关功能基因的筛选与鉴定[D].硕士学位论文,华南农业大学,导师:江青艳,pp.19-20.(Yu K F.2010.Screening and identification of related functions and comparison of porcine muscle fatty acid composition of different varieties of gene[D].Thesis for M.S.,South China Agricultural University,Suppervisor:Jiang Q Y,pp.19-20.)
张静静,宋玉芹,李桢,等.2013.中国西门塔尔牛SCD1基因多态性与肉质性状的相关分析[J].华北农学报,28(1):54-57.(Zhang J J,Song Y Q,Li Z,et al.2013.Polymorphisms of SCD1 gene and its association with meat quality traits in Chinese simmental[J].Acta Agriculturae Boreali-singca,28(1):54-57.)
张宜辉,张蕊,张军,等.2014.鹅SCD1基因真核表达载体构建及在肝细胞中的表达[J].上海交通大学学报:农业科学版,32(2):5-9.(Zhang Y H,Zhang R,Zhang J,et al.2014.Construction and expression of goose SCD1gene in primary hepatocyte[J].Journal of Shanghai Jiaotong University:Agricul Turil Science,32(2):5-9.)
郑东旖,樊超楠,刘兆秋,等.2010.学龄前肥胖儿童硬脂酰辅酶A去饱和酶的变化[J].中妇幼保健,25(16):2225-2227.(Zheng D Y,Fan C N,Liu Z Q,et al.2010.Changes of stearoyl coenzylme A desaturase in preschool children with obesity[J].Maternal and Child Health Care of China,25(16):2225-2227.)
朱才业,韦光辉,李伟,等.2013.徐淮山羊SCD1基因的克隆和亚细胞定位研究及转基因小鼠的制备[J].中国农业科学,46(17):3695-3703.(Zhu C Y,Wei G H,Li W,et al.2013.Cloning of xuhuai goat SCD1 gene,subcellular localization and the preparation of transgenic mice[J].Scientia Agricultura Sinica,46(17):3695-3703.
Dodgson S J,Forster R E,Storey B T.1984.The role of carbonic anhydrase in hepatocyte metabolism[J].Annals of the New York Academy of Sciences,429(1):516-524.
Donato M T,Castell J V,Gomez-Lechon M J.1999.Characterization of drug metabolizing activities in pig hepatocytes for use in bioartificial liver devices:comparison with other hepatic cellular models[J].Journal of Hepatology,31(3):542-549.
Edge S B,Hoeg J M,Triche T,et al.1986.Cultured human hepatocytes.Evidence for metabolism of low density lipoproteins by a pathway independent of the classica low density lipoprotein receptor[J].Journal of Biological Chemistry,261(8):3800-3806.
Foretz M,Pacot C,Dugail I,et al.1999.ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose[J].Molecular and Cellular Biology19(5):3760-3768.
Huang G M,Jiang Q H,Cai C,et al.2015.SCD1 negatively regulates autophagy-induced cell death in human hepatocellular carcinoma through inactivation of the AMPKsignaling pathway[J].Cancer Letters,358(2):180-190.
Jaureguiberry M S,Tricerri M A,Sanchez S A,et al.2014Role of plasma membrane lipid composition on cellular homeostasis:Learning from cell line models expressing fatty acid desaturases[J].Acta Biochimica Et Biophysica Sinica,46(4):273-282.
Jeffcoat R,James A T.1978.The control of stearoyl-Co A desaturase by dietary linoleic acid[J].FEBS Letters,85(1)114-122.
Jump D B,Clarke S D.1999,Regulation of gene expression by dietary fat[J].Annual Review of Nutrition,19(1)63-90.
Liu M L,Mars W M,Zarnegar R.1994.Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-alpha in adult rat liver[J].European Journal of Neuroscience,19(6):1521-1527.
Matsusue K,Aibara D,Hayafuchi R,et al.2014.Hepatic PPAR gamma and LXR alpha independently regulate lipid accumulation in the livers of genetically obese mice[J].FEBS Letters,588(14):2277-2281.
Miyazaki M,Kim Y,Ntambi J.2001.A lipogenic diet in mice with a disruption of the stearoyl-Co A desaturase 1 gene reveals a stringent requirement of endogenous monounsaturated fatty acids for triglyceride synthesis[J].Journal of Lipid Research,42(7):1018-1024.
Paton C M,Ntambi J M.2009.Biochemical and physiological function of stearoyl-Co A desaturase[J].American Journal of Physiology,297(1):E28-37.
Pichard L,Raulet E,Fabre G,et al.1986.Human hepatocyte culture[J].Methods in Molecular Biology,8(1):33-50.
Ralston J C,Metherel A H,Stark K D,et al.2016.SCD1 mediates the influence of exogenous saturated and monounsaturated fatty acids in adipocytes:Effects on cellular stress,inflammatory markers and fatty acid elongation[J].Journal of Nutritional Biochemistry,27(1):241-248.
李万贵,张依裕,王单单,等.2016.鸭SCD1基因对PC3细胞脂质性状的效应分析[J].基因组学与应用生物学,35(9):2355-2360.(Li W G,Zhang Y Y,Wang D D,et al2016.Effect analysis of duck SCD1 gene on lipid traits of PC3 cells[J].Genomics and Annlied Aioloev,35(9)2355-2360.)
李卫华,杨佳欣.2014.硬脂酰辅酶A去饱和酶1与恶性肿瘤的研究进展[J].中国肿瘤临床,41(17):1131-1134(Li W H,Yang J X.2014.Development in the study of the correlation between stearoyl-coenzyme A desaturase1 and cancer progression[J].Chinese Journal of Clinica Oncology,41(17):1131-1134.)
刘三凤,任军,郭源梅,等.2008.鸡SCD和Sirt1基因的SNP搜寻及其遗传多样性分析[J].畜牧兽医学报,39(3):278-285.(Liu S F,Ren J,Guo Y M,et al.2008Identification of Single nucleotide polymorphisms of SCD and evaluation of their genetic diversities in chickens[J].Acta Veterinaria et Zootechnica Sinica,39(3)278-285.)
魏聪.2016.天府肉羊SCD1基因在肌肉中的表达与棕榈油酸和油酸含量的相关性研究[D].硕士学位论文,四川农业大学,导师:徐刚毅,pp.52-53.(Wei C.2016.Expression of the SCD1 gene and its relationship with the palmitoleic acid and oliec acid composition of tianfu goat[D].Thesis for M.S.,Sichuan Agricultural University,Suppervisor:Xu G Y,pp.52-53.)
徐雅玲,黄巨恩,梁菁,等.2016.改构型酸性成纤维细胞生长因子对紫杉醇诱导体外大鼠肝细胞损伤保护作用的体外实验研究[J].暨南大学学报(自然科学与医学版),37(1):44-48.((Xu Y L,Huang J E,Liang J,et al.2016.Protective effects of Ma FGF on the injury of hepatocytes induced by taxol in vitro[J].Journal of Jinan University(Natural Science and Medicine edition),37(1):44-48.)
余凯凡.2010.不同品种猪肌肉脂肪酸组成的比较及相关功能基因的筛选与鉴定[D].硕士学位论文,华南农业大学,导师:江青艳,pp.19-20.(Yu K F.2010.Screening and identification of related functions and comparison of porcine muscle fatty acid composition of different varieties of gene[D].Thesis for M.S.,South China Agricultural University,Suppervisor:Jiang Q Y,pp.19-20.)
张静静,宋玉芹,李桢,等.2013.中国西门塔尔牛SCD1基因多态性与肉质性状的相关分析[J].华北农学报,28(1):54-57.(Zhang J J,Song Y Q,Li Z,et al.2013.Polymorphisms of SCD1 gene and its association with meat quality traits in Chinese simmental[J].Acta Agriculturae Boreali-singca,28(1):54-57.)
张宜辉,张蕊,张军,等.2014.鹅SCD1基因真核表达载体构建及在肝细胞中的表达[J].上海交通大学学报:农业科学版,32(2):5-9.(Zhang Y H,Zhang R,Zhang J,et al.2014.Construction and expression of goose SCD1gene in primary hepatocyte[J].Journal of Shanghai Jiaotong University:Agricul Turil Science,32(2):5-9.)
郑东旖,樊超楠,刘兆秋,等.2010.学龄前肥胖儿童硬脂酰辅酶A去饱和酶的变化[J].中妇幼保健,25(16):2225-2227.(Zheng D Y,Fan C N,Liu Z Q,et al.2010.Changes of stearoyl coenzylme A desaturase in preschool children with obesity[J].Maternal and Child Health Care of China,25(16):2225-2227.)
朱才业,韦光辉,李伟,等.2013.徐淮山羊SCD1基因的克隆和亚细胞定位研究及转基因小鼠的制备[J].中国农业科学,46(17):3695-3703.(Zhu C Y,Wei G H,Li W,et al.2013.Cloning of xuhuai goat SCD1 gene,subcellular localization and the preparation of transgenic mice[J].Scientia Agricultura Sinica,46(17):3695-3703.
Dodgson S J,Forster R E,Storey B T.1984.The role of carbonic anhydrase in hepatocyte metabolism[J].Annals of the New York Academy of Sciences,429(1):516-524.
Donato M T,Castell J V,Gomez-Lechon M J.1999.Characterization of drug metabolizing activities in pig hepatocytes for use in bioartificial liver devices:comparison with other hepatic cellular models[J].Journal of Hepatology,31(3):542-549.
Edge S B,Hoeg J M,Triche T,et al.1986.Cultured human hepatocytes.Evidence for metabolism of low density lipoproteins by a pathway independent of the classica low density lipoprotein receptor[J].Journal of Biological Chemistry,261(8):3800-3806.
Foretz M,Pacot C,Dugail I,et al.1999.ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose[J].Molecular and Cellular Biology19(5):3760-3768.
Huang G M,Jiang Q H,Cai C,et al.2015.SCD1 negatively regulates autophagy-induced cell death in human hepatocellular carcinoma through inactivation of the AMPKsignaling pathway[J].Cancer Letters,358(2):180-190.
Jaureguiberry M S,Tricerri M A,Sanchez S A,et al.2014Role of plasma membrane lipid composition on cellular homeostasis:Learning from cell line models expressing fatty acid desaturases[J].Acta Biochimica Et Biophysica Sinica,46(4):273-282.
Jeffcoat R,James A T.1978.The control of stearoyl-Co A desaturase by dietary linoleic acid[J].FEBS Letters,85(1)114-122.
Jump D B,Clarke S D.1999,Regulation of gene expression by dietary fat[J].Annual Review of Nutrition,19(1)63-90.
Liu M L,Mars W M,Zarnegar R.1994.Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-alpha in adult rat liver[J].European Journal of Neuroscience,19(6):1521-1527.
Matsusue K,Aibara D,Hayafuchi R,et al.2014.Hepatic PPAR gamma and LXR alpha independently regulate lipid accumulation in the livers of genetically obese mice[J].FEBS Letters,588(14):2277-2281.
Miyazaki M,Kim Y,Ntambi J.2001.A lipogenic diet in mice with a disruption of the stearoyl-Co A desaturase 1 gene reveals a stringent requirement of endogenous monounsaturated fatty acids for triglyceride synthesis[J].Journal of Lipid Research,42(7):1018-1024.
Paton C M,Ntambi J M.2009.Biochemical and physiological function of stearoyl-Co A desaturase[J].American Journal of Physiology,297(1):E28-37.
Pichard L,Raulet E,Fabre G,et al.1986.Human hepatocyte culture[J].Methods in Molecular Biology,8(1):33-50.
Ralston J C,Metherel A H,Stark K D,et al.2016.SCD1 mediates the influence of exogenous saturated and monounsaturated fatty acids in adipocytes:Effects on cellular stress,inflammatory markers and fatty acid elongation[J].Journal of Nutritional Biochemistry,27(1):241-248.