饲粮阴阳离子差水平对小鼠血钙浓度及胃肠道组织钙结合蛋白-D9k mRNA相对表达水平的影响
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摘要
本试验在前期建立了动物胃肠道钙代谢相关基因表达水平检测体系与表达谱分析的基础上,旨在进一步确定饲粮阴阳离子差(DCAD)水平对动物血钙浓度和胃肠道组织钙结合蛋白-D9k(Ca BP-D9k)mRNA相对表达水平的影响,为揭示低DCAD水平防治动物低血钙症的作用机制提供依据。将120只昆明小鼠随机分为3组,每组40只,自配种前3 d起分别饲喂DCAD水平为+300(高DCAD水平组,HD组)、+150(对照组,CON组)、-50(低DCAD水平组,LD组)的饲粮。检测母鼠产前20 d(-20 d)、产前5 d(-5 d)、产后当天(0 d)、产后3 d(+3 d)血钙浓度和胃肠道组织Ca BP-D9k mRNA相对表达水平。结果表明,与HD组相比,LD组显著提高了围产期内0 d、+3 d小鼠血钙浓度(P<0.05),显著上调了小肠肠段(十二指肠、空肠、回肠)与结肠Ca BP-D9k mRNA相对表达水平(P<0.05),这一效应在-5 d、0 d表现最为显著(P<0.05),并在+3 d提高了小鼠空肠与结肠的Ca BP-D9k mRNA相对表达水平(P<0.05)。统计结果显示,十二指肠、空肠与结肠3个位点的Ca BP-D9k mRNA相对表达水平与DCAD水平、血钙浓度具有显著的关联性(P<0.05)。由此可见,降低DCAD水平可上调动物小肠及结肠肠段Ca BP-D9k mRNA相对表达水平,同时伴随更高的血钙浓度。这可能是低DCAD水平有效维持动物围产期血钙稳恒,降低低血钙发生率的重要途径。
Based on the calcium related gene expression profile and its analysis,the study was conducted to investigate the effects of dietary cation-anion difference( DCAD) level on plasma calcium concentration and gastrointestinal tract tissues calcium binding proteins-D9k( Ca BP-D9k) relative expression level of mice during the transition period,and to clarify the mechanism of low DCAD level prevent hypocalcaemia. One hundred and tw enty KMmice were randomly allotted into 3 groups with 40 mice in each group,mice in the 3 groups were fed 3 diets with DCAD level at + 300( high DCAD level group,HD group),+ 150( control group,CON group),and- 150( low DCAD level group,LD group),respectively. Plasma calcium concentration and gastrointestinal tract tissues Ca BP-D9 k mRNA relative expression level were detected at 4 time points: day of 20(- 20 d) and 5(- 5 d) before kidding,day of kidding( 0 d),and day of 3 after kidding( + 3 d). The results showed that compared with the HD group,the plasma calcium concentration of mice at 0 d and + 3 d during the transition period in LD group was significantly increased( P< 0.05),and the Ca BP-D9 k mRNA relative expression level was significantly up-regulated in duodenum,jejunum,ileum and colon( P< 0.05),especially observed on- 5 d before parturition and 0 d at parturition( P < 0.05). Also,Ca BP-D9 k mRNA relative expression level was significantly increased in jejunum and colon on the + 3 d after parturition( P< 0.05). Moreover,there was a significant correlation betw een Ca BP-D9 k mRNA relative expression level in duodenum,jejunum and colon and plasma calcium concentration. In conclusion,low DCAD level is able to up-regulate Ca BP-D9 k mRNA relative expression level in small intestine and colon,accompany with greater plasma calcium concentration. This is possibly an important way of low DCAD level maintains plasma calcium homeostasis during the transition period,and reduce the occurrence rate of hypocalcemia.
Based on the calcium related gene expression profile and its analysis,the study was conducted to investigate the effects of dietary cation-anion difference( DCAD) level on plasma calcium concentration and gastrointestinal tract tissues calcium binding proteins-D9k( Ca BP-D9k) relative expression level of mice during the transition period,and to clarify the mechanism of low DCAD level prevent hypocalcaemia. One hundred and tw enty KMmice were randomly allotted into 3 groups with 40 mice in each group,mice in the 3 groups were fed 3 diets with DCAD level at + 300( high DCAD level group,HD group),+ 150( control group,CON group),and- 150( low DCAD level group,LD group),respectively. Plasma calcium concentration and gastrointestinal tract tissues Ca BP-D9 k mRNA relative expression level were detected at 4 time points: day of 20(- 20 d) and 5(- 5 d) before kidding,day of kidding( 0 d),and day of 3 after kidding( + 3 d). The results showed that compared with the HD group,the plasma calcium concentration of mice at 0 d and + 3 d during the transition period in LD group was significantly increased( P< 0.05),and the Ca BP-D9 k mRNA relative expression level was significantly up-regulated in duodenum,jejunum,ileum and colon( P< 0.05),especially observed on- 5 d before parturition and 0 d at parturition( P < 0.05). Also,Ca BP-D9 k mRNA relative expression level was significantly increased in jejunum and colon on the + 3 d after parturition( P< 0.05). Moreover,there was a significant correlation betw een Ca BP-D9 k mRNA relative expression level in duodenum,jejunum and colon and plasma calcium concentration. In conclusion,low DCAD level is able to up-regulate Ca BP-D9 k mRNA relative expression level in small intestine and colon,accompany with greater plasma calcium concentration. This is possibly an important way of low DCAD level maintains plasma calcium homeostasis during the transition period,and reduce the occurrence rate of hypocalcemia.
引文
[1]GOFF J P,HORST R L,MUELLER F J,et al.Addition of chloride to a prepartal diet high in cations increases 1,25-dihydroxyvitamin D response to hypocalcemia preventing milk fever[J].Journal of Dairy Science,1991,74(11):3863-3871.
[2]曹国凡.甲状旁腺素和钙在促进骨形成中的协同作用及机制研究[D].博士学位论文.南京:南京医科大学,2008.
[3]WU W X,LIU J X,XU G Z,et al.Calcium homeostasis,acid-base balance,and health status in periparturient Holstein cow s fed diets w ith low cation-anion difference[J].Livestock Science,2008,117(1):7-14.
[4]APPER-BOSSARD E,FAVERDIN P,Meschy F,et al.Effects of dietary cation-anion difference on ruminal metabolism and blood acid-base regulation in dairy cow s receiving 2 contrasting levels of concentrate in diets[J].Journal of Dairy Science,2010,93(9):4196-4210.
[5]MARTN-TERESO J,VERSTEGEN M W A.A novel model to explain dietary factors affecting hypocalcaemia in dairy cattle[J].Nutrition Research Review s,2011,24(2):228-243.
[6]GOFF J P.Macromineral physiology and application to the feeding of the dairy cow for prevention of milk fever and other periparturient mineral disorders[J].Animal Feed Science and Technology,2006,126(3/4):237-257.
[7]BERNE R M,LEVY M N,KOEPPEN B M,et al.Berne&levy physiology[M].Philadelphia,PA:M osby/Elsevier,2008.
[8]MARTN-TERESO LPEZ J.Rumen-protected rice bran to induce the adaptation of calcium metabolism in dairy cow s[D].Ph.D.Thesis.Wageningen:Wageningen University,2010.
[9]THILSING-HANSEN T,JRGENSEN R J,STERGAARD S.Milk fever control principles:a review[J].Acta Veterinaria Scandinavica,2002,43(1):1-19.
[10]GROSS M,KUMAR R.Physiology and biochemistry of vitamin D-dependent calcium binding proteins[J].The American Journal of Physiology,1990,259(2 Pt2):F195-209.
[11]毛萌,王道聪.VD及Ca BP-Ds在肠道和肾脏钙转运中的作用[J].国外医学:儿科学分册,1996,23(6):289-292.
[12]杨艺,莘海亮,夏先林,等.昆明小鼠胃肠道钙离子跨膜吸收途径相关基因表达模式分析[J].动物营养学报,2013,25(2):441-446.
[13]刘和娣.钙结合蛋白研究进展[J].国外医学生理、病理科学与临床分册,1995(4):278-281.
[14]REPLOGLE R A,LI Q,WANG L B,et al.Gene-bydiet interactions influence calcium absorption and bone density in mice[J].Journal of Bone and M ineral Research,2014,29(3):657-665.
[15]莘海亮,吴文旋,李胜利.动物胃肠道钙吸收关键基因表达实时荧光定量PCR检测方法的建立[J].江苏农业科学,2013,41(4):31-34.
[16]WILLEMS E,LEYNS L,VANDESOMPELE J.Standardization of real-time PCR gene expression data from independent biological replicates[J].Analytical Biochemistry,2008,379(1):127-129.
[17]BOLT M J G,CAO L P,KONG J,et al.Vitamin D receptor is required for dietary calcium-induced repression of calbindin-D9k expression in mice[J].The Journal of Nutritional Biochemistry,2005,16(5):286-290.
[18]MULLEN P A.Clinical and biochemical responses to the treatment of milk fever[J].Veterinary Record,1975,97(5):87-92.
[19]HORST R L,GOFF J P,REINHARDT T A.Adapting to the transition betw een gestation and lactation:differences betw een rat,human and dairy cow[J].Journal of M ammary Gland Biology and Neoplasia,2005,10(2):141-156.
[20]WILKENS M R,OBERHEIDE I,SCHRDER B,et al.Influence of the combination of 25-hydroxyvitamin D3and a diet negative in cation-anion difference on peripartal calcium homeostasis of dairy cow s[J].Journal of Dairy Science,2012,95(1):151-164.
[21]董淑筠,伍晓倩,赵俊红.孕期母体血钙变化及544例孕妇血钙分析[J].复旦学报:医学版,2002,29(5):418-419.
[22]LEAN I J,DEGARIS P J,Mcneil D M,et al.Hypocalcemia in dairy cow s:meta-analysis and dietary cation anion difference theory revisited[J].Journal of Dairy Science,2006,89(2):669-684.
[23]LIESEGANG A.Influence of anionic salts on bone metabolism in periparturient dairy goats and sheep[J].Journal of Dairy Science,2008,91(6):2449-2460.
[24]HIRATA Y,OKU Y.TRP channels are involved in mediating hypercapnic Ca2+responses in rat glia-rich medullary cultures independent of extracellular p H[J].Cell Calcium,2010,48(2/3):124-132.
[25]PREZ A V,PICOTTO G,CARPENTIERI A R,et al.M inireview on regulation of intestinal calcium absorption.Emphasis on molecular mechanisms of transcellular pathw ay[J].Digestion,2008,77(1):22-34.
[26]YAMAGISHI N,MIYAZAKI M,NAITO Y.The expression of genes for transepithelial calcium-transporting proteins in the bovine duodenum[J].The Veterinary Journal,2006,171(2):363-366.
[27]SCHRDER B,BREVES G.Mechanisms and regulation of calcium absorption from the gastrointestinal tract in pigs and ruminants:comparative aspects w ith special emphasis on hypocalcemia in dairy cow s[J].Animal Health Research Review s,2006,7(1/2):31-41.
[28]BRUN L R,BRANCE M L,RIGALLI A.Luminal calcium concentration controls intestinal calcium absorption by modification of intestinal alkaline phosphatase activity[J].British Journal of Nutrition,2012,108(2):229-233.
[29]VANOEVELEN J,JANSSENS A,HUITEMA L F A,et al.Trpv5/6 is vital for epithelial calcium uptake and bone formation[J].The FASEB Journal,2011,25(9):3197-3207.
[2]曹国凡.甲状旁腺素和钙在促进骨形成中的协同作用及机制研究[D].博士学位论文.南京:南京医科大学,2008.
[3]WU W X,LIU J X,XU G Z,et al.Calcium homeostasis,acid-base balance,and health status in periparturient Holstein cow s fed diets w ith low cation-anion difference[J].Livestock Science,2008,117(1):7-14.
[4]APPER-BOSSARD E,FAVERDIN P,Meschy F,et al.Effects of dietary cation-anion difference on ruminal metabolism and blood acid-base regulation in dairy cow s receiving 2 contrasting levels of concentrate in diets[J].Journal of Dairy Science,2010,93(9):4196-4210.
[5]MARTN-TERESO J,VERSTEGEN M W A.A novel model to explain dietary factors affecting hypocalcaemia in dairy cattle[J].Nutrition Research Review s,2011,24(2):228-243.
[6]GOFF J P.Macromineral physiology and application to the feeding of the dairy cow for prevention of milk fever and other periparturient mineral disorders[J].Animal Feed Science and Technology,2006,126(3/4):237-257.
[7]BERNE R M,LEVY M N,KOEPPEN B M,et al.Berne&levy physiology[M].Philadelphia,PA:M osby/Elsevier,2008.
[8]MARTN-TERESO LPEZ J.Rumen-protected rice bran to induce the adaptation of calcium metabolism in dairy cow s[D].Ph.D.Thesis.Wageningen:Wageningen University,2010.
[9]THILSING-HANSEN T,JRGENSEN R J,STERGAARD S.Milk fever control principles:a review[J].Acta Veterinaria Scandinavica,2002,43(1):1-19.
[10]GROSS M,KUMAR R.Physiology and biochemistry of vitamin D-dependent calcium binding proteins[J].The American Journal of Physiology,1990,259(2 Pt2):F195-209.
[11]毛萌,王道聪.VD及Ca BP-Ds在肠道和肾脏钙转运中的作用[J].国外医学:儿科学分册,1996,23(6):289-292.
[12]杨艺,莘海亮,夏先林,等.昆明小鼠胃肠道钙离子跨膜吸收途径相关基因表达模式分析[J].动物营养学报,2013,25(2):441-446.
[13]刘和娣.钙结合蛋白研究进展[J].国外医学生理、病理科学与临床分册,1995(4):278-281.
[14]REPLOGLE R A,LI Q,WANG L B,et al.Gene-bydiet interactions influence calcium absorption and bone density in mice[J].Journal of Bone and M ineral Research,2014,29(3):657-665.
[15]莘海亮,吴文旋,李胜利.动物胃肠道钙吸收关键基因表达实时荧光定量PCR检测方法的建立[J].江苏农业科学,2013,41(4):31-34.
[16]WILLEMS E,LEYNS L,VANDESOMPELE J.Standardization of real-time PCR gene expression data from independent biological replicates[J].Analytical Biochemistry,2008,379(1):127-129.
[17]BOLT M J G,CAO L P,KONG J,et al.Vitamin D receptor is required for dietary calcium-induced repression of calbindin-D9k expression in mice[J].The Journal of Nutritional Biochemistry,2005,16(5):286-290.
[18]MULLEN P A.Clinical and biochemical responses to the treatment of milk fever[J].Veterinary Record,1975,97(5):87-92.
[19]HORST R L,GOFF J P,REINHARDT T A.Adapting to the transition betw een gestation and lactation:differences betw een rat,human and dairy cow[J].Journal of M ammary Gland Biology and Neoplasia,2005,10(2):141-156.
[20]WILKENS M R,OBERHEIDE I,SCHRDER B,et al.Influence of the combination of 25-hydroxyvitamin D3and a diet negative in cation-anion difference on peripartal calcium homeostasis of dairy cow s[J].Journal of Dairy Science,2012,95(1):151-164.
[21]董淑筠,伍晓倩,赵俊红.孕期母体血钙变化及544例孕妇血钙分析[J].复旦学报:医学版,2002,29(5):418-419.
[22]LEAN I J,DEGARIS P J,Mcneil D M,et al.Hypocalcemia in dairy cow s:meta-analysis and dietary cation anion difference theory revisited[J].Journal of Dairy Science,2006,89(2):669-684.
[23]LIESEGANG A.Influence of anionic salts on bone metabolism in periparturient dairy goats and sheep[J].Journal of Dairy Science,2008,91(6):2449-2460.
[24]HIRATA Y,OKU Y.TRP channels are involved in mediating hypercapnic Ca2+responses in rat glia-rich medullary cultures independent of extracellular p H[J].Cell Calcium,2010,48(2/3):124-132.
[25]PREZ A V,PICOTTO G,CARPENTIERI A R,et al.M inireview on regulation of intestinal calcium absorption.Emphasis on molecular mechanisms of transcellular pathw ay[J].Digestion,2008,77(1):22-34.
[26]YAMAGISHI N,MIYAZAKI M,NAITO Y.The expression of genes for transepithelial calcium-transporting proteins in the bovine duodenum[J].The Veterinary Journal,2006,171(2):363-366.
[27]SCHRDER B,BREVES G.Mechanisms and regulation of calcium absorption from the gastrointestinal tract in pigs and ruminants:comparative aspects w ith special emphasis on hypocalcemia in dairy cow s[J].Animal Health Research Review s,2006,7(1/2):31-41.
[28]BRUN L R,BRANCE M L,RIGALLI A.Luminal calcium concentration controls intestinal calcium absorption by modification of intestinal alkaline phosphatase activity[J].British Journal of Nutrition,2012,108(2):229-233.
[29]VANOEVELEN J,JANSSENS A,HUITEMA L F A,et al.Trpv5/6 is vital for epithelial calcium uptake and bone formation[J].The FASEB Journal,2011,25(9):3197-3207.