油酸和亚油酸对山羊乳腺细胞甘油三酯含量及乳脂合成相关基因表达的影响
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摘要
为探讨外源添加油酸和亚油酸对体外培养的奶山羊乳腺上皮细胞甘油三酯(TG)含量和乳脂肪合成相关基因表达的影响,试验以奶山羊乳腺上皮细胞为研究对象,分别用0、50、100、200μmol/L油酸和0、20、40、80μmol/L的亚油酸处理细胞24h,检测细胞内TG的含量;采用实时荧光定量PCR法检测细胞内乳脂合成相关基因mRNA水平的变化。结果显示,100、200μmol/L的油酸和40、80μmol/L亚油酸均显著促进细胞内TG的合成(P<0.05)。实时荧光定量PCR结果表明,添加100、200μmol/L油酸可显著上调二酰甘油酰基转移酶2(DGAT2)、乙酰辅酶A羧化酶(ACC)及脂肪酸合成酶(FASN)基因的表达(P<0.05),显著抑制硬脂酰辅酶A去饱和酶1(SCD1)基因的表达(P<0.05),而对过氧化物酶体增殖物激活受体γ(PPARγ)基因的表达无显著影响(P>0.05)。添加20μmol/L亚油酸可显著抑制ACC和FASN基因的表达(P<0.05);亚油酸浓度为40和80μmol/L时可显著上调DGAT2基因的表达(P<0.05),抑制SCD1和SREBP1基因的表达(P<0.05),对ACC、FASN和PPARγ基因表达均无显著影响(P>0.05)。综上所述,100～200μmol/L油酸和40～80μmol/L亚油酸对奶山羊乳腺上皮细胞乳脂肪合成具有较好的促进效果。
The aim of this study was to investigate the effects of oleic acid and linoleic acid on triglyceride(TG)content and the expression of genes involved in milk fat synthesis in dairy goat mammary epithelial cells in vitro.The goat mammary epithelial cells were used as the research object,and different concentrations of oleic acid(0,50,100,200μmol/L)and linoleic acid(0,20,40,80μmol/L)were added in culture medium for a 24 hcultivation.The intracellular TG content was measured by using triglyceride determination kit,and gene expressions were detected by Realtime quantitative PCR.The results showed that 100,200μmol/L oleic acid and 40,80μmol/L linoleic acid significantly increased TG content(P<0.05).The expression of DGAT2,ACC andFASNgenes were significantly up-regulated by addition of 100 and 200μmol/L oleic acid(P<0.05),while the SCD1 gene expression was significantly inhibited by oleic acid(P<0.05).However,oleic acid had no significant effect on the expression of PPARγgene(P>0.05).The results of linoleic acid addition showed that 20μmol/L linoleic acid could significantly inhibit the expressions of ACCand FASNgenes(P<0.05).40 and 80μmol/L linoleic acid could significantly promote the expression of DGAT2 gene(P<0.05),inhibit the expression of SCD1 and SREBP1 genes(P<0.05),and had no significant effect on the expression of ACC,FASN and PPARγgenes(P>0.05).In conclusion,100 to 200μmol/L oleic acid and 40 to 80μmol/L linoleic acid were optimal levels considering its improvement effects on milk fat synthesis in dairy goat mammary epithelial cells.
The aim of this study was to investigate the effects of oleic acid and linoleic acid on triglyceride(TG)content and the expression of genes involved in milk fat synthesis in dairy goat mammary epithelial cells in vitro.The goat mammary epithelial cells were used as the research object,and different concentrations of oleic acid(0,50,100,200μmol/L)and linoleic acid(0,20,40,80μmol/L)were added in culture medium for a 24 hcultivation.The intracellular TG content was measured by using triglyceride determination kit,and gene expressions were detected by Realtime quantitative PCR.The results showed that 100,200μmol/L oleic acid and 40,80μmol/L linoleic acid significantly increased TG content(P<0.05).The expression of DGAT2,ACC andFASNgenes were significantly up-regulated by addition of 100 and 200μmol/L oleic acid(P<0.05),while the SCD1 gene expression was significantly inhibited by oleic acid(P<0.05).However,oleic acid had no significant effect on the expression of PPARγgene(P>0.05).The results of linoleic acid addition showed that 20μmol/L linoleic acid could significantly inhibit the expressions of ACCand FASNgenes(P<0.05).40 and 80μmol/L linoleic acid could significantly promote the expression of DGAT2 gene(P<0.05),inhibit the expression of SCD1 and SREBP1 genes(P<0.05),and had no significant effect on the expression of ACC,FASN and PPARγgenes(P>0.05).In conclusion,100 to 200μmol/L oleic acid and 40 to 80μmol/L linoleic acid were optimal levels considering its improvement effects on milk fat synthesis in dairy goat mammary epithelial cells.
引文
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[16]BONNET M,BERNARD L,BES S,et al.Selection of reference genes for quantitative Real-time PCR normalisation in adipose tissue,muscle,liver and mammary gland from ruminants[J].Animal,2013,7(8):1344-1353.
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[18]BERNARD L,LEROUX C,CHILLIARD Y.Expression and nutritional regulation of lipogenic genes in the ruminant lactating mammary gland[J].Advances in Experimental Medicine and Biology,2008,606(1):67-108.
[19]邢媛媛,李红磊,李大彪,等.油酸对奶牛乳腺上皮细胞内甘油三酯含量和乳脂肪合成相关基因表达的影响[J].中国畜牧杂志,2017,53(7):56-60.XING Y Y,LI H L,LI D B,et al.Effects of oleic acid on triglyceride content and gene expressions involved in milk fat synthesis in bovine mammary epithelial cells[J].Chinese Journal of Animal Science,2017,53(7):56-60.(in Chinese)
[20]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.
[21]YAO D W,LUO J,HE Q Y,et al.Liver X receptorαpromotes the synthesis of monounsaturated fatty acids in goat mammary epithelial cells via the control of stearoyl-coenzyme A desaturase 1in an SREBP-1-dependent manner[J].Journal of Dairy Science,2016,99(8):6391-6402.
[22]YAO D W,LUO J,HE Q Y,et al.SCD1alters longchain fatty acid(LCFA)composition and its expression is directly regulated by SREBP-1and PPARγin dairy goat mammary cells[J].Journal of Cellular Physiology,2016,232(3):635-649.
[23]SHI H B,LUO J,YAO D W,et al.Peroxisome proliferator-activated receptor-gamma stimulates the synthesis of monounsaturated fatty acids in dairy goat mammary epithelial cells via the control of stearoylcoenzyme A desaturase[J].Journal of Dairy Science,2013,96(12):7844-7853.
[24]王乐山,左旭冬,江青艳,等.脂肪酸对动物骨骼肌的代谢调控作用[J].中国畜牧兽医,2017,44(3):748-754.WANG L S,ZUO X D,JIANG Q Y,et al.The metabolic regulatory effect of fatty acid on mammal skeletal muscle[J].China Animal Husbandry&Veterinary Medicine,2017,44(3):748-754.(in Chinese)
[25]HARVATINE K,BAUMAN D.SREBP1and thyroid hormone responsive spot 14(S14)are involved in the regulation of bovine mammary lipid synthesis during diet-induced milk fat depression and treatment with CLA[J].Journal of Nutrition,2006,136(10):2468-2474.
[2]BERNARD L,ROUEL J,LEROUX C,et al.Mammary lipid metabolism and milk fatty acid secretion in Alpine goats fed vegetable lipids[J].Journal of Dairy Science,2005,88(4):1478-1489.
[3]MATHER I H,KEENAN T W.Origin and secretion of milk lipids[J].Journal of Mammary Gland Biology and Neoplasia,1998,3(3):259-273.
[4]YONEZAWA T,YONEKURA S,KOBAYASHI Y,et al.Effects of long chain fatty acids on cytosolic triacylglycerol accumulation and lipid droplet formation in primary cultured bovine mammary epithelial cells[J].Journal of Dairy Science,2004,87(8):2527-2534.
[5]BIONAZ M,LOOR J J.Gene networks driving bovine milk fat synthesis during the lactation cycle[J].BMCGenomics,2008,9(1):366.
[6]YU K,SHI H B,LUO J,et al.PPARG modulated lipid accumulation in dairy GMEC via regulation of ADRPgene[J].Journal of Cellular Biochemistry,2015,116(1):192-201.
[7]SHI H B,LUO J,ZHU J,et al.PPAR gamma regulates genes involved in triacylglycerol synthesis and secretion in mammary gland epithelial cells of dairy goats[J].PPAR Research,2013,2013(6):310948.
[8]XU H F,LUO J,ZHAO W S,et al.Overexpression of SREBP1(sterol regulatory element binding protein 1)promotes de novo fatty acid synthesis and triacylglycerol accumulation in goat mammary epithelial cells[J].Journal of Dairy Science,2016,99(1):783-795.
[9]崔瑞莲,王加启,卜登攀,等.不同十八碳脂肪酸对奶牛乳腺上皮细胞增殖及甘油三酯合成的影响[J].畜牧兽医学报,2012,43(7):1064-1070.CUI R L,WANG J Q,BU D P,et al.Effects of 18-carbon fatty acid on cell proliferation and triacylylycerol accumulation in bovine mammary gland epithelial cells in vitro[J].Acta Veterinaria et Zootechnica Sinica,2012,43(7):1064-1070.(in Chinese)
[10]孙晓菊.十八碳不饱和脂肪酸对乳腺上皮细胞脂肪代谢的影响及其机制研究[D].呼和浩特:内蒙古农业大学,2012.SUN X J.Effects of C18unasturated fatty acids on lipid metabolism and its mechanisms in mammary epithelial cells[D].Hohhot:Inner Mongolia Agricultural University,2012.(in Chinese)
[11]王桢,罗军,王伟,等.奶山羊乳腺上皮细胞的分离、培养及鉴定[J].生物工程学报,2010,26(8):1123-1127.WANG Z,LUO J,WANG W,et al.Characterization and culture of isolated primary dairy goat mammary gland epithelial cells[J].Chinese Journal of Biotechnology,2010,26(8):1123-1127.(in Chinese)
[12]SHI H P,SHI H B,LUO J,et al.Establishment and characterization of a dairy goat mammary epithelial cell line with human telomerase(hT-MECs)[J].Animal Science Journal,2014,85(7):735-743.
[13]李君,权凯,张桂枝,等.催乳激素对山羊脂肪酸合酶基因转录活性的影响[J].中国畜牧兽医,2018,45(8):2255-2262.LI J,QUAN K,ZHANG G Z,et al.Effect of lactogenic hormones on the transcriptional activity of FASNgene in goats[J].China Animal Husbandry&Veterinary Medicine,2018,45(8):2255-2262.(in Chinese)
[14]梁梦瑶,高学军,侯晓明,等.油酸和硬脂酸对奶牛乳腺上皮细胞中脂肪酸结合蛋白3表达和脂滴分泌的影响[J].中国畜牧兽医,2014,41(5):137-141.LIANG M Y,GAO X J,HOU X M,et al.Effect of oleic acid and stearic acid on the expression of fatty acid bingding protein 3and secretion of lipid droplets in dairy cow mammary epithelial cells[J].China Animal Husbandry&Veterinary Medicine,2014,41(5):137-141.(in Chinese)
[15]BIONAZ M,LOOR J J.Identification of reference genes for quantitative Real-time PCR in the bovine mammary gland during the lactation cycle[J].Physiology Genomics,2007,29(3):312-319.
[16]BONNET M,BERNARD L,BES S,et al.Selection of reference genes for quantitative Real-time PCR normalisation in adipose tissue,muscle,liver and mammary gland from ruminants[J].Animal,2013,7(8):1344-1353.
[17]FOX P F,MCSWEENEY P L H.Advanced dairy chemistry:Lipids[M].3rd Edition.New York:Springer,2006.
[18]BERNARD L,LEROUX C,CHILLIARD Y.Expression and nutritional regulation of lipogenic genes in the ruminant lactating mammary gland[J].Advances in Experimental Medicine and Biology,2008,606(1):67-108.
[19]邢媛媛,李红磊,李大彪,等.油酸对奶牛乳腺上皮细胞内甘油三酯含量和乳脂肪合成相关基因表达的影响[J].中国畜牧杂志,2017,53(7):56-60.XING Y Y,LI H L,LI D B,et al.Effects of oleic acid on triglyceride content and gene expressions involved in milk fat synthesis in bovine mammary epithelial cells[J].Chinese Journal of Animal Science,2017,53(7):56-60.(in Chinese)
[20]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.
[21]YAO D W,LUO J,HE Q Y,et al.Liver X receptorαpromotes the synthesis of monounsaturated fatty acids in goat mammary epithelial cells via the control of stearoyl-coenzyme A desaturase 1in an SREBP-1-dependent manner[J].Journal of Dairy Science,2016,99(8):6391-6402.
[22]YAO D W,LUO J,HE Q Y,et al.SCD1alters longchain fatty acid(LCFA)composition and its expression is directly regulated by SREBP-1and PPARγin dairy goat mammary cells[J].Journal of Cellular Physiology,2016,232(3):635-649.
[23]SHI H B,LUO J,YAO D W,et al.Peroxisome proliferator-activated receptor-gamma stimulates the synthesis of monounsaturated fatty acids in dairy goat mammary epithelial cells via the control of stearoylcoenzyme A desaturase[J].Journal of Dairy Science,2013,96(12):7844-7853.
[24]王乐山,左旭冬,江青艳,等.脂肪酸对动物骨骼肌的代谢调控作用[J].中国畜牧兽医,2017,44(3):748-754.WANG L S,ZUO X D,JIANG Q Y,et al.The metabolic regulatory effect of fatty acid on mammal skeletal muscle[J].China Animal Husbandry&Veterinary Medicine,2017,44(3):748-754.(in Chinese)
[25]HARVATINE K,BAUMAN D.SREBP1and thyroid hormone responsive spot 14(S14)are involved in the regulation of bovine mammary lipid synthesis during diet-induced milk fat depression and treatment with CLA[J].Journal of Nutrition,2006,136(10):2468-2474.