RNA干扰DGAT1和DGAT2在猪肝细胞和肌内前体脂肪细胞中表达的研究
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摘要
本研究旨在探索DGAT1和DGAT2基因表达对猪脂肪沉积效率的影响。以3日龄宗地花猪为实验动物,针对DGAT1和DGAT2基因m RNA序列设计6对sh RNA序列,构建RNA干扰表达载体,将DGAT1和DGAT2的sh RNA干扰载体分别转染至猪的肝细胞和肌内前体脂肪细胞中,采用RT-q PCR筛选出肝细胞和肌内前体脂肪细胞中的最佳sh RNA干扰载体,将最佳干扰载体再次转染肝细胞和肌内前体脂肪细胞,测定细胞代谢液中甘油三酯和总胆固醇含量变化。结果表明:所构建的干扰载体对肝细胞中DGAT1和DGAT2基因的最高抑制效率分别为78.45%和87.52%,对肌内前体脂肪细胞中DGAT1和DGAT2基因的最高抑制效率分别为82.24%和89.82%;肝细胞和肌内前体脂肪细胞培养液中甘油三酯和总胆固醇的含量均降低,其中转染p GPU6/GFP/Neo-DGAT2-1实验组的肝细胞培养液中甘油三酯含量显著低于空白组(P<0.05)。结果提示,抑制DGAT1和DGAT2基因的表达,可降低猪肝细胞和肌内前体脂肪细胞代谢液中甘油三酯和总胆固醇的含量。
The purpose of this study was to explore the effect of DGAT1 and DGAT2 genes expression on fat deposition efficiency in pigs. In this study, 6 pairs of sh RNA sequences were designed for m RNA sequences of DGAT1 and DGAT2 genes, and RNA interference expression vectors were constructed by used 3 days old Zongdihua pigs as experimental animals, the sh RNA interference vectors of DGAT1 and DGAT2 were transfected into porcine hepatocytes and intramuscular precursor adipocytes respectively, the best sh RNA interference vectors in hepatocytes and intramuscular precursor adipocytes were selected by RT-q PCR retransfection of optimal interference vectors into Hepatocytes and Intramuscular Precursor Adipocytes, triglyceride and total cholesterol contents changes were tested in cells metabolic fluid. The results indicated that the maximum inhibitory efficiency of the constructed interference vectors on DGAT1 and DGAT2 genes in Hepatocytes was 78.45% and 87.52%, respectively, and that on DGAT1 and DGAT2 genes in intramuscular precursor adipocytes was 82.24% and 89.82%, respectively, the contents of triglyceride and total cholesterol in Hepatocytes and Intramuscular precursor adipocytes were decreased in metabolic fluid. The contents of triglyceride in hepatocytes metabolic fluid transfected with p GPU6/GFP/Neo-DGAT2-1 group was significantly lower than that of blank group(P<0.05). Inhibiting the expression of DGAT1 and DGAT2 genes could decrease the contents of triglyceride and total cholesterol in the metabolites of porcine Hepatocytes and Intramuscular Precursor Adipocytes.
The purpose of this study was to explore the effect of DGAT1 and DGAT2 genes expression on fat deposition efficiency in pigs. In this study, 6 pairs of sh RNA sequences were designed for m RNA sequences of DGAT1 and DGAT2 genes, and RNA interference expression vectors were constructed by used 3 days old Zongdihua pigs as experimental animals, the sh RNA interference vectors of DGAT1 and DGAT2 were transfected into porcine hepatocytes and intramuscular precursor adipocytes respectively, the best sh RNA interference vectors in hepatocytes and intramuscular precursor adipocytes were selected by RT-q PCR retransfection of optimal interference vectors into Hepatocytes and Intramuscular Precursor Adipocytes, triglyceride and total cholesterol contents changes were tested in cells metabolic fluid. The results indicated that the maximum inhibitory efficiency of the constructed interference vectors on DGAT1 and DGAT2 genes in Hepatocytes was 78.45% and 87.52%, respectively, and that on DGAT1 and DGAT2 genes in intramuscular precursor adipocytes was 82.24% and 89.82%, respectively, the contents of triglyceride and total cholesterol in Hepatocytes and Intramuscular precursor adipocytes were decreased in metabolic fluid. The contents of triglyceride in hepatocytes metabolic fluid transfected with p GPU6/GFP/Neo-DGAT2-1 group was significantly lower than that of blank group(P<0.05). Inhibiting the expression of DGAT1 and DGAT2 genes could decrease the contents of triglyceride and total cholesterol in the metabolites of porcine Hepatocytes and Intramuscular Precursor Adipocytes.
引文
[1]Chen H C,Farese R V.Inhibition of triglyceride synthesis as a treatment strategy for obesity lessons from DGAT1-deficient mice[J].Arterioscl Throm Vas,2005,25(3):482-486.
[2]Liu L,Shi X,Choi C S,et al.Paradoxical coupling of triglyceride synthesis and fatty acid oxidation in skeletal muscle overexpressing DGAT1[J].Diabetes,2009,58(11):2516-2524.
[3]Buhman K K,Smith S J,Stone S J,et al.DGAT1 is not essential for intestinal triacylglycerol absorption or chylomicron synthesis[J].J Biol Chem,2002,277(28):25474-25479.
[4]Stone S J,Levin M C,Farese R V.Membrane topology and identification of key functional amino acid residues of murine acyl-CoA:diacylglycerol acyltransferase-2[J].J Biol Chem,2006,281(52):40273-40282.
[5]McFie P J,Stone S L,Banman S L,et al.Topological orientation of acyl-CoA:diacylglycerol acyltransferase-1(DGAT1)and identification of a putative active site histidine and the role of the n terminus in dimer/tetramer formation[J].JBiol Chem,2010,285(48):37377-37387.
[6]Qi J,Lang W,Giardino E,et al.High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity[J].J Lipid Res,2010,51(12):3559-3567.
[7]Renaville B,Bacciu N,Lanzoni M,et al.Polymorphism of fat metabolism genes as candidate markers for meat quality and production traits in heavy pigs[J].Meat Sci,2015,110:220-223.
[8]胡悦,李标,王继英,等.猪背膘组织二脂酰甘油酰基转移酶基因(DGAT1和DGAT2)的发育性表达分析[J].农业生物技术学报,2010,18(5):905-910.
[9]Harris C A,Haas J T,Streeper R S,et al.DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes[J].J Lipid Res,2011,52(4):657-667.
[10]王国富,高树新,张立凡,等.大白猪二脂酰甘油酰基转移酶1和2基因(DGAT1和DGAT2)发育性表达特点及其与背膘厚的关联分析[J].农业生物技术学报,2011(4):705-710.
[11]Chen H C,Farese R V.Inhibition of triglyceride synthesis as a treatment strategy for obesity lessons from DGAT1-deficient mice[J].Arterioscl Throm Vas,2005,25(3):482-486.
[12]Yu Y H,Ginsberg H.The role of acyl-CoA:diacylglycerol acyltransferase(DGAT)in energy metabolism[J].Ann Med,2004,36(4):252-261.
[13]Suzuki R,Tobe K,Aoyama M,et al.Expression of DGAT2 in white adipose tissue is regulated by central leptin action[J].JBiol Chem,2005,280(5):3331-3337.
[14]Stone S J,Myers H M,Watkins S M,et al.Lipopenia and skin barrier abnormalities in DGAT2-deficient mice[J].J Biol Chem,2004,279(12):11767-11776.
[15]Chen H C,Smith S J,Tow B,et al.Leptin modulates the effects of acyl CoA:diacylglycerol acyltransferase deficiency on murine fur and sebaceous glands[J].J Clin Invest,2002,109(2):175-268.
[16]Li D W,Cen S Y,Liu Y H,et al.A type 2 diacylglycerol acyltransferase accelerates the triacylglycerol biosynthesis in heterokont oleaginous microalga Nannochloropsis oceanica[J].J Biotechnol,2016,229:65-71.
[17]Smith S J,Cases S,Jensen D R,et al.Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat[J].Nat Genet,2000,25(1):87-90.
[2]Liu L,Shi X,Choi C S,et al.Paradoxical coupling of triglyceride synthesis and fatty acid oxidation in skeletal muscle overexpressing DGAT1[J].Diabetes,2009,58(11):2516-2524.
[3]Buhman K K,Smith S J,Stone S J,et al.DGAT1 is not essential for intestinal triacylglycerol absorption or chylomicron synthesis[J].J Biol Chem,2002,277(28):25474-25479.
[4]Stone S J,Levin M C,Farese R V.Membrane topology and identification of key functional amino acid residues of murine acyl-CoA:diacylglycerol acyltransferase-2[J].J Biol Chem,2006,281(52):40273-40282.
[5]McFie P J,Stone S L,Banman S L,et al.Topological orientation of acyl-CoA:diacylglycerol acyltransferase-1(DGAT1)and identification of a putative active site histidine and the role of the n terminus in dimer/tetramer formation[J].JBiol Chem,2010,285(48):37377-37387.
[6]Qi J,Lang W,Giardino E,et al.High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity[J].J Lipid Res,2010,51(12):3559-3567.
[7]Renaville B,Bacciu N,Lanzoni M,et al.Polymorphism of fat metabolism genes as candidate markers for meat quality and production traits in heavy pigs[J].Meat Sci,2015,110:220-223.
[8]胡悦,李标,王继英,等.猪背膘组织二脂酰甘油酰基转移酶基因(DGAT1和DGAT2)的发育性表达分析[J].农业生物技术学报,2010,18(5):905-910.
[9]Harris C A,Haas J T,Streeper R S,et al.DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes[J].J Lipid Res,2011,52(4):657-667.
[10]王国富,高树新,张立凡,等.大白猪二脂酰甘油酰基转移酶1和2基因(DGAT1和DGAT2)发育性表达特点及其与背膘厚的关联分析[J].农业生物技术学报,2011(4):705-710.
[11]Chen H C,Farese R V.Inhibition of triglyceride synthesis as a treatment strategy for obesity lessons from DGAT1-deficient mice[J].Arterioscl Throm Vas,2005,25(3):482-486.
[12]Yu Y H,Ginsberg H.The role of acyl-CoA:diacylglycerol acyltransferase(DGAT)in energy metabolism[J].Ann Med,2004,36(4):252-261.
[13]Suzuki R,Tobe K,Aoyama M,et al.Expression of DGAT2 in white adipose tissue is regulated by central leptin action[J].JBiol Chem,2005,280(5):3331-3337.
[14]Stone S J,Myers H M,Watkins S M,et al.Lipopenia and skin barrier abnormalities in DGAT2-deficient mice[J].J Biol Chem,2004,279(12):11767-11776.
[15]Chen H C,Smith S J,Tow B,et al.Leptin modulates the effects of acyl CoA:diacylglycerol acyltransferase deficiency on murine fur and sebaceous glands[J].J Clin Invest,2002,109(2):175-268.
[16]Li D W,Cen S Y,Liu Y H,et al.A type 2 diacylglycerol acyltransferase accelerates the triacylglycerol biosynthesis in heterokont oleaginous microalga Nannochloropsis oceanica[J].J Biotechnol,2016,229:65-71.
[17]Smith S J,Cases S,Jensen D R,et al.Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat[J].Nat Genet,2000,25(1):87-90.