鸭LXRα基因过表达对原代肝细胞及PC3细胞脂质代谢的效应研究
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摘要
旨在探究鸭LXRα基因对脂肪代谢的表达调控机制,本试验以pEGFP-N3为载体,构建携带HIS标签的LXRα基因真核过表达载体,并将过表达载体分别转染鸭原代肝细胞及PC3细胞,使用鸭HIS标签试剂盒测定LXRα基因在肝细胞及PC3细胞中的过表达量,分析该基因分别在两种细胞中过表达与各脂质指标含量的相关性,进而探究其对鸭肝细胞及PC3细胞脂质代谢的调控作用。试验结果显示,LXRα基因在两种细胞中过表达与三酰甘油含量(triglyceride,TG)及高密度脂蛋白(high density lipoprotein,HDL)含量均呈极显著正相关关系(P<0.01),与总胆固醇(total cholesterol,TC)含量及低密度脂蛋白含量(low density lipoprotein,LDL)则均呈极显著负相关关系(P<0.01)。本研究揭示了LXRα基因过表达对TG和HDL的合成代谢有重要的正向调控作用,对TC和LDL具有反向调控作用,且在这两种类型的细胞中调控机制相似。这一研究结果将对今后深入研究LXRα基因在脂质代谢网络通路中的作用及解决鸭体脂肪过度沉积等问题奠定理论基础。
To explore the regulation mechanism of duck LXRαgene on fat deposition,in this experiment,the pEGFP-N3 was used as the vector to construct the LXRαgene eukaryotic overexpression vector carried HIS-tag that was transfected into the duck primary hepatocytes and PC3 cells,respectively.The over expression level of LXRαgene in duck hepatocytes and PC3 cells were detected by the duck HIS-tag kit,and the correlation between the over expression of LXRαgene in the two kinds of cells and the lipid index contents was analyzed,and the regulation mechanism of LXRαgene on lipid metabolism in duck hepatocytes and PC3 cells was explored.The results showed that the over expression of LXRαgene in both cells were significantly positively correlated with the contents of triglyceride(TG)and high-density lipoprotein(HDL)(P<0.01),and significantly negatively correlated with the contents of total cholesterol(TC)and low density lipoprotein(LDL)(P<0.01).The results reveal that over expression of LXRαgene has a positive regulatory effect on the anabolism of TG and HDL,and has a negative regulatory effect on TC and LDL,and the regulatory mechanisms in the two types of cells are similar.The results of this study will lay a theoretical foundation for the further study of the role of LXRαgene in the lipid metabolism network pathway and the solve of excessive fat deposition in duck body.
To explore the regulation mechanism of duck LXRαgene on fat deposition,in this experiment,the pEGFP-N3 was used as the vector to construct the LXRαgene eukaryotic overexpression vector carried HIS-tag that was transfected into the duck primary hepatocytes and PC3 cells,respectively.The over expression level of LXRαgene in duck hepatocytes and PC3 cells were detected by the duck HIS-tag kit,and the correlation between the over expression of LXRαgene in the two kinds of cells and the lipid index contents was analyzed,and the regulation mechanism of LXRαgene on lipid metabolism in duck hepatocytes and PC3 cells was explored.The results showed that the over expression of LXRαgene in both cells were significantly positively correlated with the contents of triglyceride(TG)and high-density lipoprotein(HDL)(P<0.01),and significantly negatively correlated with the contents of total cholesterol(TC)and low density lipoprotein(LDL)(P<0.01).The results reveal that over expression of LXRαgene has a positive regulatory effect on the anabolism of TG and HDL,and has a negative regulatory effect on TC and LDL,and the regulatory mechanisms in the two types of cells are similar.The results of this study will lay a theoretical foundation for the further study of the role of LXRαgene in the lipid metabolism network pathway and the solve of excessive fat deposition in duck body.
引文
[1]ZHANG Y,BREEVOORT S R,ANGDISEN J,et al.Liver LXRαexpression is crucial for whole body cholesterol homeostasis and reverse cholesterol transport in mice[J].J Clin Invest,2012,122(5):1688-1699.
[2]HOZOJI M,MUNEHIRA Y,IKEDA Y,et al.Direct interaction of nuclear liver X receptor-βwith ABCA1modulates cholesterol efflux[J].J Biol Chem,2008,283(44):30057-30063.
[3]WRANGE O,OKRET S,RADOJC′IC′M,et al.Characterization of the purified activated glucocorticoid receptor from rat liver cytosol[J].J Biol Chem,1984,259(7):4534-4541.
[4]MCKENNA N J,O′MALLEY B W.Combinatorial control of gene expression by nuclear receptors and coregulators[J].Cell,2002,108(4):465-474.
[5]ZHAO Y P,LI L,MA J P,et al.LXRαgene downregulation by lentiviral-based RNA interference enhances liver function after fatty liver transplantation in rats[J].Hepatobiliary Pancreat Dis Int,2015,14(4):386-393.
[6]GE C X,YU R,XU M X,et al.Betaine prevented fructose-induced NAFLD by regulating LXRα/PPARαpathway and alleviating ER stress in rats[J].Eur J Pharmacol,2016,770:154-164.
[7]LI Y Y,LI X C,SUN W K,et al.Comparison of liver microRNA transcriptomes of Tibetan and Yorkshire pigs by deep sequencing[J].Gene,2016,577(2):244-250.
[8]郑芬萍.LXRs激动对PPARγ转录调节脂肪因子表达的作用及机制研究[D].杭州:浙江大学,2011.ZHENG F P.The effect of LXRs activation on the expression of adipokines transcriptional regulated by PPARγand its possible mechanisms[D].Hangzhou:Zhejiang University,2011.(in Chinese)
[9]GREFHORST A,PARKS E J.Reduced insulin-mediated inhibition of VLDL secretion upon pharmacological activation of the liver X receptor in mice[J].JLipid Res,2009,50(7):1374-1383.
[10]BOVENGAF,SABBC,MOSCHETTA A.Uncoupling nuclear receptor LXR and cholesterol metabolism in cancer[J].Cell Metab,2015,21(4):517-526.
[11]REMEN K M R,GUSTAFSSON J,ANDERSSONG.The liver X receptor promotes macrophage differentiation and suppresses osteoclast formation in mouse RAW264.7promyelocytic leukemia cells exposed to bacterial lipopolysaccharide[J].Biochem Biophys Res Commun,2013,430(1):375-380.
[12]LAURENCIKIENE J,RYDN M.Liver X receptors and fat cell metabolism[J].Int J Obes(Lond),2012,36(12):1494-1502.
[13]BISCHOFF E D,DAIGE C L,PETROWSKI M,et al.Non-redundant roles for LXRαand LXRβin atherosclerosis susceptibility in low density lipoprotein receptor knockout mice[J].J Lipid Res,2010,51(5):900-906.
[14]罗华伦,李万贵,张依裕,等.鸭SCD1基因过表达对原代肝细胞脂质代谢的效应研究[J].农业生物技术学报,2018,26(3):476-483.LUO H L,LI W G,ZHANG Y Y,et al.Effects of duck(Anas platyrhynchos)SCD1gene over-expression on lipid metabolism in primary hepatocytes[J].Journal of Agricultural Biotechnology,2018,26(3):476-483.(in Chinese)
[15]WANG B,TONTONOZ P.Liver X receptors in lipid signalling and membrane homeostasis[J].Nat Rev Endocrinol,2018,14(8):452-463.
[16]李涛,向蕾.肝X受体-胆固醇逆转运与炎症的共同平台[J].中国动脉硬化杂志,2014,22(4):417-420.LI T,XIANG L.Liver X receptors:the common platform of reverse cholesterol transport and inflammation[J].Chinese Journal of Arteriosclerosis,2014,22(4):417-420.(in Chinese)
[17]TAKAHASHI Y,ITO Y,WADA N,et al.Development of homogeneous assay for simultaneous measurement of apoE-deficient,apoE-containing,and total HDL-cholesterol[J].Clin Chim Acta,2016,454:135-142.
[18]KASHIWAGI K,YANAGIDA M,MATSUI D,et al.Expression of liver X receptors in normal and refractory carcinoma tissues of the human lung and pancreas[J].Histol Histopathol,2018,33(5):497-505.
[19]张依裕,李万贵,陈彦希,等.鸭LXRα基因表达与肉质和血清生化指标的相关性[J].贵州农业科学,2014,42(7):116-119.ZHANG Y Y,LI W G,CHEN Y X,et al.Correlation analysis between LXRαgene expression and meat quality and serum biochemical levels on ducks[J].Guizhou Agricultural Sciences,2014,42(7):116-119.(in Chinese)
[20]WANG Y J,ROGERS P M,SU C,et al.Regulation of cholesterologenesis by the oxysterol receptor,LXRα[J].J Biol Chem,2008,283(39):26332-26339.
[21]代小艳,唐朝克.肝X受体在体内胆固醇平衡中的作用[J].中国病理生理杂志,2006,22(9):1854-1857.DAI X X,TANG C K.The role of liver X receptors in the control of cholesterol homeostasis[J].Chinese Journal of Pathophysiology,2006,22(9):1854-1857.(in Chinese)
[22]LENGI A J,CORL B A.Short communication:identification of the bovine sterol regulatory element binding protein-1cpromoter and its activation by liver Xreceptor[J].J Dairy Sci,2010,93(12):5831-5836.
[23]OPPI-WILLIAMS C,SUAGEE J K,CORL B A.Regulation of lipid synthesis by liver X receptorαand sterol regulatory element-binding protein 1in mammary epithelial cells[J].J Dairy Sci,2013,96(1):112-121.
[24]LI J,LUO J,XU H F,et al.Fatty acid synthase promoter:characterization,and transcriptional regulation by sterol regulatory element binding protein-1in goat mammary epithelial cells[J].Gene,2015,561(1):157-164.
[25]OKAZAKI H,GOLDSTEIN J L,BROWN M S,et al.LXR-SREBP-1c-phospholipid transfer protein axis controls very low density lipoprotein(VLDL)particle size[J].J Biol Chem,2010,285(9):6801-6810.
[26]许会芬.SREBP-1基因对山羊乳腺上皮细胞脂肪酸代谢的调控作用研究[D].杨凌:西北农林科技大学,2016.XU H F.The regulatory function of SREBP-1gene on fatty acid metabolism in goat mammary gland epithelial cells[D].Yangling:Northwest A&F University,2016.(in Chinese)
[27]戴爽,颜元良,龚志成.SCD1抑制剂抗肿瘤作用的研究进展[J].中国现代医学杂志,2018,28(16):52-58.DAI S,YAN Y L,GONG Z C.Progression of SCD1inhibitor as an antitumor reagent for human cancers[J].China Journal of Modern Medicine,2018,28(16):52-58.(in Chinese)
[28]黄光明,沈薇.SCD1对肝癌SMMC-7721细胞脂质代谢的影响及可能机制[J].肿瘤学杂志,2017,23(8):653-657.HUANG G M,SHEN W.Effects of SCD1on lipid metabolism in SMMC-7721cells and its underlying molecular mechanism[J].Journal of Chinese Oncology,2017,23(8):653-657.(in Chinese)
[29]李万贵,张依裕,王单单,等.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,2016,35(9):2355-2360.LI W G,ZHANG Y Y,WANG D D,et al.Effect analysis of duck SCD1gene on lipid traits of PC3cells[J].Genomics and Applied Biology,2016,35(9):2355-2360.(in Chinese)
[2]HOZOJI M,MUNEHIRA Y,IKEDA Y,et al.Direct interaction of nuclear liver X receptor-βwith ABCA1modulates cholesterol efflux[J].J Biol Chem,2008,283(44):30057-30063.
[3]WRANGE O,OKRET S,RADOJC′IC′M,et al.Characterization of the purified activated glucocorticoid receptor from rat liver cytosol[J].J Biol Chem,1984,259(7):4534-4541.
[4]MCKENNA N J,O′MALLEY B W.Combinatorial control of gene expression by nuclear receptors and coregulators[J].Cell,2002,108(4):465-474.
[5]ZHAO Y P,LI L,MA J P,et al.LXRαgene downregulation by lentiviral-based RNA interference enhances liver function after fatty liver transplantation in rats[J].Hepatobiliary Pancreat Dis Int,2015,14(4):386-393.
[6]GE C X,YU R,XU M X,et al.Betaine prevented fructose-induced NAFLD by regulating LXRα/PPARαpathway and alleviating ER stress in rats[J].Eur J Pharmacol,2016,770:154-164.
[7]LI Y Y,LI X C,SUN W K,et al.Comparison of liver microRNA transcriptomes of Tibetan and Yorkshire pigs by deep sequencing[J].Gene,2016,577(2):244-250.
[8]郑芬萍.LXRs激动对PPARγ转录调节脂肪因子表达的作用及机制研究[D].杭州:浙江大学,2011.ZHENG F P.The effect of LXRs activation on the expression of adipokines transcriptional regulated by PPARγand its possible mechanisms[D].Hangzhou:Zhejiang University,2011.(in Chinese)
[9]GREFHORST A,PARKS E J.Reduced insulin-mediated inhibition of VLDL secretion upon pharmacological activation of the liver X receptor in mice[J].JLipid Res,2009,50(7):1374-1383.
[10]BOVENGAF,SABBC,MOSCHETTA A.Uncoupling nuclear receptor LXR and cholesterol metabolism in cancer[J].Cell Metab,2015,21(4):517-526.
[11]REMEN K M R,GUSTAFSSON J,ANDERSSONG.The liver X receptor promotes macrophage differentiation and suppresses osteoclast formation in mouse RAW264.7promyelocytic leukemia cells exposed to bacterial lipopolysaccharide[J].Biochem Biophys Res Commun,2013,430(1):375-380.
[12]LAURENCIKIENE J,RYDN M.Liver X receptors and fat cell metabolism[J].Int J Obes(Lond),2012,36(12):1494-1502.
[13]BISCHOFF E D,DAIGE C L,PETROWSKI M,et al.Non-redundant roles for LXRαand LXRβin atherosclerosis susceptibility in low density lipoprotein receptor knockout mice[J].J Lipid Res,2010,51(5):900-906.
[14]罗华伦,李万贵,张依裕,等.鸭SCD1基因过表达对原代肝细胞脂质代谢的效应研究[J].农业生物技术学报,2018,26(3):476-483.LUO H L,LI W G,ZHANG Y Y,et al.Effects of duck(Anas platyrhynchos)SCD1gene over-expression on lipid metabolism in primary hepatocytes[J].Journal of Agricultural Biotechnology,2018,26(3):476-483.(in Chinese)
[15]WANG B,TONTONOZ P.Liver X receptors in lipid signalling and membrane homeostasis[J].Nat Rev Endocrinol,2018,14(8):452-463.
[16]李涛,向蕾.肝X受体-胆固醇逆转运与炎症的共同平台[J].中国动脉硬化杂志,2014,22(4):417-420.LI T,XIANG L.Liver X receptors:the common platform of reverse cholesterol transport and inflammation[J].Chinese Journal of Arteriosclerosis,2014,22(4):417-420.(in Chinese)
[17]TAKAHASHI Y,ITO Y,WADA N,et al.Development of homogeneous assay for simultaneous measurement of apoE-deficient,apoE-containing,and total HDL-cholesterol[J].Clin Chim Acta,2016,454:135-142.
[18]KASHIWAGI K,YANAGIDA M,MATSUI D,et al.Expression of liver X receptors in normal and refractory carcinoma tissues of the human lung and pancreas[J].Histol Histopathol,2018,33(5):497-505.
[19]张依裕,李万贵,陈彦希,等.鸭LXRα基因表达与肉质和血清生化指标的相关性[J].贵州农业科学,2014,42(7):116-119.ZHANG Y Y,LI W G,CHEN Y X,et al.Correlation analysis between LXRαgene expression and meat quality and serum biochemical levels on ducks[J].Guizhou Agricultural Sciences,2014,42(7):116-119.(in Chinese)
[20]WANG Y J,ROGERS P M,SU C,et al.Regulation of cholesterologenesis by the oxysterol receptor,LXRα[J].J Biol Chem,2008,283(39):26332-26339.
[21]代小艳,唐朝克.肝X受体在体内胆固醇平衡中的作用[J].中国病理生理杂志,2006,22(9):1854-1857.DAI X X,TANG C K.The role of liver X receptors in the control of cholesterol homeostasis[J].Chinese Journal of Pathophysiology,2006,22(9):1854-1857.(in Chinese)
[22]LENGI A J,CORL B A.Short communication:identification of the bovine sterol regulatory element binding protein-1cpromoter and its activation by liver Xreceptor[J].J Dairy Sci,2010,93(12):5831-5836.
[23]OPPI-WILLIAMS C,SUAGEE J K,CORL B A.Regulation of lipid synthesis by liver X receptorαand sterol regulatory element-binding protein 1in mammary epithelial cells[J].J Dairy Sci,2013,96(1):112-121.
[24]LI J,LUO J,XU H F,et al.Fatty acid synthase promoter:characterization,and transcriptional regulation by sterol regulatory element binding protein-1in goat mammary epithelial cells[J].Gene,2015,561(1):157-164.
[25]OKAZAKI H,GOLDSTEIN J L,BROWN M S,et al.LXR-SREBP-1c-phospholipid transfer protein axis controls very low density lipoprotein(VLDL)particle size[J].J Biol Chem,2010,285(9):6801-6810.
[26]许会芬.SREBP-1基因对山羊乳腺上皮细胞脂肪酸代谢的调控作用研究[D].杨凌:西北农林科技大学,2016.XU H F.The regulatory function of SREBP-1gene on fatty acid metabolism in goat mammary gland epithelial cells[D].Yangling:Northwest A&F University,2016.(in Chinese)
[27]戴爽,颜元良,龚志成.SCD1抑制剂抗肿瘤作用的研究进展[J].中国现代医学杂志,2018,28(16):52-58.DAI S,YAN Y L,GONG Z C.Progression of SCD1inhibitor as an antitumor reagent for human cancers[J].China Journal of Modern Medicine,2018,28(16):52-58.(in Chinese)
[28]黄光明,沈薇.SCD1对肝癌SMMC-7721细胞脂质代谢的影响及可能机制[J].肿瘤学杂志,2017,23(8):653-657.HUANG G M,SHEN W.Effects of SCD1on lipid metabolism in SMMC-7721cells and its underlying molecular mechanism[J].Journal of Chinese Oncology,2017,23(8):653-657.(in Chinese)
[29]李万贵,张依裕,王单单,等.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,2016,35(9):2355-2360.LI W G,ZHANG Y Y,WANG D D,et al.Effect analysis of duck SCD1gene on lipid traits of PC3cells[J].Genomics and Applied Biology,2016,35(9):2355-2360.(in Chinese)