亮氨酸对猪胎盘滋养层细胞增殖及氨基酸转运的影响
|
摘要
为研究亮氨酸(Leu)对猪胎盘滋养层细胞(pTr)增殖、凋亡以及氨基酸转运载体表达的影响及其机制,本试验用不同浓度Leu(0、1、10 mmol/L)分别处理pTr细胞24 h和48 h后,使用荧光定量PCR技术检测pTr细胞增殖和凋亡相关基因、氨基酸转运载体以及mTOR信号通路关键蛋白等的mRNA表达水平。结果表明:Leu处理pTr细胞24 h后,1 mmol/L试验组的SNAT1(P<0.01)、4E-BP1 (P<0.05)和eIF4G(P<0.05)的mRNA相对表达量低于对照组;Leu处理pTr细胞48 h后,1 mmol/L试验组LAT1(P<0.05)、4E-BP1(P<0.01)的mRNA相对表达量低于对照组,10 mmol/L试验组CDK4(P<0.05)、4E-BP1 (P <0.01)、SNAT1 (P <0.01)、SNAT2 (P <0.01)、LAT1 (P <0.01)以及rBAT (P <0.05)的mRNA相对表达量也低于对照组;Leu处理pTr细胞24 h和48 h后,10 mmol/L组mTORC1的mRNA相对表达量较对照组和1 mmol/L组均极显著提高(P<0.01)。可见,10 mmol/L Leu会抑制pTr细胞的增殖活力,并可能通过mTOR信号通路的介导,降低了pTr细胞氨基酸转运载体的表达。
To investigate the effects and mechanisms of leucine on proliferation, apoptosis and regulation of amino acid transporters in porcine trophectoderm cells(pTr), pTr cells were treated with different concentrations of Leu(0, 1, 10 mmol/L) for 24 h and 48 h in this experiment. The mRNA of pTr cell proliferation and apoptosis-related genes, amino acid transporter, the key proteins in mTOR signaling pathway were detected by real-time PCR. The results showed that the relative expression levels of SNAT1(P<0.01), 4 E-BP1(P<0.05) and eIF4 G(P<0.05) in the 1 mmol/L test group were lower than those in the control group after treatment with Leu for 24 h. After treatment of pTr cells for 48 h, the relative expression levels of LAT1(P<0.05) and 4 EBP1(P<0.01) in the 1 mmol/L test group were lower than those in the control group, and 10 mmol/L test group CDK4(P<0.05). 4 E-BP1(P<0.01), SNAT1(P<0.01), SNAT2(P<0.01), LAT1(P<0.01), and rBAT(P<0.05) mRNA expression levels were also lower than the control group; Leu treatment After 24 h and 48 h of pTr cells, the relative expression of mTORC1 mRNA in 10 mmol/L group was significantly higher than that in the control group and 1 mmol/L group(P<0.01). All the currently available data demonstrate that 10 mmol/L Leu can suppress the proliferation of pTr and reduces the expression of amino acid transporters in pTr cells by affecting the mTOR signaling pathway.
To investigate the effects and mechanisms of leucine on proliferation, apoptosis and regulation of amino acid transporters in porcine trophectoderm cells(pTr), pTr cells were treated with different concentrations of Leu(0, 1, 10 mmol/L) for 24 h and 48 h in this experiment. The mRNA of pTr cell proliferation and apoptosis-related genes, amino acid transporter, the key proteins in mTOR signaling pathway were detected by real-time PCR. The results showed that the relative expression levels of SNAT1(P<0.01), 4 E-BP1(P<0.05) and eIF4 G(P<0.05) in the 1 mmol/L test group were lower than those in the control group after treatment with Leu for 24 h. After treatment of pTr cells for 48 h, the relative expression levels of LAT1(P<0.05) and 4 EBP1(P<0.01) in the 1 mmol/L test group were lower than those in the control group, and 10 mmol/L test group CDK4(P<0.05). 4 E-BP1(P<0.01), SNAT1(P<0.01), SNAT2(P<0.01), LAT1(P<0.01), and rBAT(P<0.05) mRNA expression levels were also lower than the control group; Leu treatment After 24 h and 48 h of pTr cells, the relative expression of mTORC1 mRNA in 10 mmol/L group was significantly higher than that in the control group and 1 mmol/L group(P<0.01). All the currently available data demonstrate that 10 mmol/L Leu can suppress the proliferation of pTr and reduces the expression of amino acid transporters in pTr cells by affecting the mTOR signaling pathway.
引文
[1]董书圣,田亮,颜培实.猪胎盘绒毛滋养层细胞体外原代培养[J].畜牧兽医学报,2017,48(1):75-82.
[2]黄璇.IGF-1对大鼠胎盘滋养层细胞增殖和GLUTs mRNA表达的影响[D].广州:华南农业大学,2010.
[3]Grillo M A,Lanza A,Colombatto S.Transport of amino acids through the placenta and their role[J].Amino Acids,2008,34(4):517-523.
[4]Cramer S,Beveridge M,Kilberg M,et al.Physiological importance of system A-mediated amino acid transport to rat fetal development[J].Amer J Phy-Cell Phy,2002,282(l):C153-160.
[5]Dicke J M,Henderson G I.Placental amino acid uptake in normal and complicated pregnancies[J].Am J Med Sci,1988,295(3):223-227.
[6]Kavitha J V,Rosario F J,Nijland M J,et al.Down-regulation of placental mTOR,insulin/IGF-I signaling,and nutrient transporters in response to maternal nutrient restriction in the baboon[J].FASEB J,2014,28(3):1294-1305.
[7]刘炀,王旭贞,管武太,等.亮氨酸对猪胎盘滋养层细胞增殖和周期的影响[J].中国畜牧杂志,2016,52(7):35-40.
[8]Falco M D,Fedele V,Cobellis L,et al..Pattern of expression of cycli n D1/CDK4 complex i n hu man placent a du r i ng gestation[J].Cell Tis Res,2004,317(2):187-194.
[9]Takeuchi S,Nswashiro H,Wada K,et al.L-Leucine induces growth arrest and persistent ERK activation in glioma cells[J].Amino Acids,2012,43(2):717-724.
[10]Kerr J F,Wyllie A H,Currie A R.Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics[J].Bri J Cancer,1972,26(4):239-257.
[11]Steller H.Mechanisms and genes of cellular suicide[J].Science,1995,267(5203):1445-1449.
[12]Meier P,Finch A,Evan G.Apoptosis in development[J].Nature,2000,407(6805):796-801.
[13]李敏,林俊.细胞凋亡途径及其机制[J].国际妇产科学杂志,2014,41(2):103-107.
[14]冯波.海芒果种子提取物对人肝癌HepG2细胞增殖、周期和凋亡的影响及其作用机制[D].上海:第二军医大学,2009.
[15]Wakshlag J J,Kallfelz F A,Wakahlag R R.The effects of branched-chain amino acids on canine neoplastic cell proliferation and death[J].J Nutr,2006,136(Suppl):2007s-2010s.
[16]Wilson F A,Suryawan A,Orellana R A,et al..Differential effects of long-term leucine infusion on tissue protein synthesis in neonatal pigs[J].Amino acids,2011,40(1):157-165.
[17]王超先,王旭贞,管武太,等.母猪妊娠后期日粮添加亮氨酸对断奶仔猪生长性能及乳成分的影响[J].中国畜牧杂志,2016,52(9):48-52.
[18]彭飞.日粮能量水平对妊娠SD大鼠胎盘印记基因表达及胎儿生长发育的影响[D].成都:四川农业大学,2012.
[19]Zhang S,Ren M,Zeng X,et al.Leucine stimulates ASCT2 amino acid transporter expression in porcine jejunal epithelial cell line(IPEC-J2)through PI3K/Akt/mTOR and ERK signaling pathways[J].Amino Acids,2014,46(12):2633-2642.
[20]Edinger A L,Thompson C B.Akt maintains cell size and survival by increasing mTOR-dependent nutrient uptake[J].Mol Biol Cell,2002,13(7):2276-2288.
[21]Liu X M,Reyna S V,Ensenat D,et al.Platelet-derived growth factor stimulates LAT1 gene expression in vascular smooth muscle:role in cell growth[J].FASEB J,2004,18(6):768-770.
[22]Luo J,Chen D,Yu B,et al.Upregulation of amino acid transporter expression induced by L-leucine availability in L6myotubes is associated with ATF4 signaling through mTORC1-dependent mechanism[J].Nutrition,2013,29(1):284-290.
[23]Escobar J,Frank J W,Suryawan A,et al.Physiological rise in plasma leucine stimulates muscle protein synthesis in neonatal pigs by enhancing translation initiation factor activation[J].Amer J Phy-End Meta,2005,288(5):E914-E921.
[24]Yuan H X,Xiong Y,Guan K L.Nutrient sensing,metabolism,and cell growth control[J].Molc Cell,2013,49(3):379-387.
[25]Wessels A G,Kluge H,Hirche F,et al.High leucine diets stimulate cerebral branched-chain amino acid degradation and modify serotonin and ketone body concentrations in a pig model[J].PLoS One,2016,11(3):e0150376.
[2]黄璇.IGF-1对大鼠胎盘滋养层细胞增殖和GLUTs mRNA表达的影响[D].广州:华南农业大学,2010.
[3]Grillo M A,Lanza A,Colombatto S.Transport of amino acids through the placenta and their role[J].Amino Acids,2008,34(4):517-523.
[4]Cramer S,Beveridge M,Kilberg M,et al.Physiological importance of system A-mediated amino acid transport to rat fetal development[J].Amer J Phy-Cell Phy,2002,282(l):C153-160.
[5]Dicke J M,Henderson G I.Placental amino acid uptake in normal and complicated pregnancies[J].Am J Med Sci,1988,295(3):223-227.
[6]Kavitha J V,Rosario F J,Nijland M J,et al.Down-regulation of placental mTOR,insulin/IGF-I signaling,and nutrient transporters in response to maternal nutrient restriction in the baboon[J].FASEB J,2014,28(3):1294-1305.
[7]刘炀,王旭贞,管武太,等.亮氨酸对猪胎盘滋养层细胞增殖和周期的影响[J].中国畜牧杂志,2016,52(7):35-40.
[8]Falco M D,Fedele V,Cobellis L,et al..Pattern of expression of cycli n D1/CDK4 complex i n hu man placent a du r i ng gestation[J].Cell Tis Res,2004,317(2):187-194.
[9]Takeuchi S,Nswashiro H,Wada K,et al.L-Leucine induces growth arrest and persistent ERK activation in glioma cells[J].Amino Acids,2012,43(2):717-724.
[10]Kerr J F,Wyllie A H,Currie A R.Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics[J].Bri J Cancer,1972,26(4):239-257.
[11]Steller H.Mechanisms and genes of cellular suicide[J].Science,1995,267(5203):1445-1449.
[12]Meier P,Finch A,Evan G.Apoptosis in development[J].Nature,2000,407(6805):796-801.
[13]李敏,林俊.细胞凋亡途径及其机制[J].国际妇产科学杂志,2014,41(2):103-107.
[14]冯波.海芒果种子提取物对人肝癌HepG2细胞增殖、周期和凋亡的影响及其作用机制[D].上海:第二军医大学,2009.
[15]Wakshlag J J,Kallfelz F A,Wakahlag R R.The effects of branched-chain amino acids on canine neoplastic cell proliferation and death[J].J Nutr,2006,136(Suppl):2007s-2010s.
[16]Wilson F A,Suryawan A,Orellana R A,et al..Differential effects of long-term leucine infusion on tissue protein synthesis in neonatal pigs[J].Amino acids,2011,40(1):157-165.
[17]王超先,王旭贞,管武太,等.母猪妊娠后期日粮添加亮氨酸对断奶仔猪生长性能及乳成分的影响[J].中国畜牧杂志,2016,52(9):48-52.
[18]彭飞.日粮能量水平对妊娠SD大鼠胎盘印记基因表达及胎儿生长发育的影响[D].成都:四川农业大学,2012.
[19]Zhang S,Ren M,Zeng X,et al.Leucine stimulates ASCT2 amino acid transporter expression in porcine jejunal epithelial cell line(IPEC-J2)through PI3K/Akt/mTOR and ERK signaling pathways[J].Amino Acids,2014,46(12):2633-2642.
[20]Edinger A L,Thompson C B.Akt maintains cell size and survival by increasing mTOR-dependent nutrient uptake[J].Mol Biol Cell,2002,13(7):2276-2288.
[21]Liu X M,Reyna S V,Ensenat D,et al.Platelet-derived growth factor stimulates LAT1 gene expression in vascular smooth muscle:role in cell growth[J].FASEB J,2004,18(6):768-770.
[22]Luo J,Chen D,Yu B,et al.Upregulation of amino acid transporter expression induced by L-leucine availability in L6myotubes is associated with ATF4 signaling through mTORC1-dependent mechanism[J].Nutrition,2013,29(1):284-290.
[23]Escobar J,Frank J W,Suryawan A,et al.Physiological rise in plasma leucine stimulates muscle protein synthesis in neonatal pigs by enhancing translation initiation factor activation[J].Amer J Phy-End Meta,2005,288(5):E914-E921.
[24]Yuan H X,Xiong Y,Guan K L.Nutrient sensing,metabolism,and cell growth control[J].Molc Cell,2013,49(3):379-387.
[25]Wessels A G,Kluge H,Hirche F,et al.High leucine diets stimulate cerebral branched-chain amino acid degradation and modify serotonin and ketone body concentrations in a pig model[J].PLoS One,2016,11(3):e0150376.