与脐带间充质干细胞共培养促进奶牛乳腺上皮细胞乳蛋白合成及其机制
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摘要
目的研究脐带间充质干细胞(UCMSC)调节奶牛乳腺上皮细胞(BMEC)乳蛋白合成的可能作用机制。方法利用TranswellTM小室将UCMSC和BMEC进行双层共培养,BMEC单纯培养为对照组,胰岛素样生长因子1受体(IGF-1R)抑制剂AG1024处理细胞,ELISA检测上清IGF-1和β酪蛋白(CSN2)、κ酪蛋白(CSN3)含量变化,实时定量PCR检测Janus激酶2/信号转导子与转录激活子5(JAK2/STAT5)信号通路相关基因的相对表达丰度;再用JAK2信号通路阻断剂AG490孵育细胞,实时定量PCR检测CSN2、CSN3 mRNA的相对表达丰度。结果与UCMSC共培养后,BMEC的CSN2、CSN3合成量和CSN2、CSN3、JAK2、STAT5、E74样ETS转录因子5(ELF5)mRNA相对表达丰度均显著高于单纯培养的BMEC;AG1024处理后,显著降低BMEC的CSN2、CSN3合成量,显著降低CSN2、CSN3、JAK2、STAT5、ELF5 mRNA的相对表达丰度;给予AG490阻断后,显著降低CSN2、CSN3 mRNA相对表达丰度;在AG1024基础上,加入AG490后显著降低CSN2、CSN3 mRNA相对表达丰度。结论UCMSC能够通过IGF-1介导JAK2/STAT5信号通路,上调BMEC乳蛋白合成关键基因mRNA的表达丰度,促进其乳蛋白的合成。
Objective To explore the possible mechanism of umbilical cord mesenchymal stem cels( UCMSCs) regulating milk protein synthesis in bovine mammary gland epithelial cells( BMECs). Methods UCMSCs and BMECs were co-cultured by double-chamber TranswellTM,and other BMECs were cultured alone as a control group. Insulin like growth factor 1 receptor( IGF-1R) inhibitor AG1024 was used to treat cells. IGF-1,β casein( CSN2) and κ casein( CSN3) content in the supernatants were determined by ELISA; the relative expression abundance of Janus kinase and signal transducer and activator of transcription factor( JAK / STAT) signaling pathway-related genes were detected by quantitative real-time PCR( qRT-PCR); and after the cells were treated with JAK2 signal blocker AG490,qRT-PCR was performed to test the relative expression abundance of CSN2 and CSN3 mRNAs. Results CSN2, CSN3 synthetic quantity, and the relative expression abundance of CSN2,CSN3,JAK2,STAT5,E74-like ETS transcription factor 5( ELF5) mRNAs of the co-culture group were significantly higher than those of the control group. After treated with AG1024,the co-culture group showed remarkably decreased CSN2,CSN3 synthesis in BMECs as wel as the decreased relative expression abundance of CSN2,CSN3,JAK2,STAT5,ELF5 mRNAs. After blocked with AG490,the relative expression abundance of CSN2 and CSN3 mRNAs were reduced significantly in the co-culture group. In addition,the expressions of CSN2 and CSN3 mRNAs were inhibited significantly when both AG1024 and AG490 were supplemented. Conclusion UCMSCs can mediate JAK2 / STAT5 signaling pathwayvia IGF-1,and increase the expressions of milk protein synthesis key genes in BMECs,thus promoteing the synthesis of milk protein.
Objective To explore the possible mechanism of umbilical cord mesenchymal stem cels( UCMSCs) regulating milk protein synthesis in bovine mammary gland epithelial cells( BMECs). Methods UCMSCs and BMECs were co-cultured by double-chamber TranswellTM,and other BMECs were cultured alone as a control group. Insulin like growth factor 1 receptor( IGF-1R) inhibitor AG1024 was used to treat cells. IGF-1,β casein( CSN2) and κ casein( CSN3) content in the supernatants were determined by ELISA; the relative expression abundance of Janus kinase and signal transducer and activator of transcription factor( JAK / STAT) signaling pathway-related genes were detected by quantitative real-time PCR( qRT-PCR); and after the cells were treated with JAK2 signal blocker AG490,qRT-PCR was performed to test the relative expression abundance of CSN2 and CSN3 mRNAs. Results CSN2, CSN3 synthetic quantity, and the relative expression abundance of CSN2,CSN3,JAK2,STAT5,E74-like ETS transcription factor 5( ELF5) mRNAs of the co-culture group were significantly higher than those of the control group. After treated with AG1024,the co-culture group showed remarkably decreased CSN2,CSN3 synthesis in BMECs as wel as the decreased relative expression abundance of CSN2,CSN3,JAK2,STAT5,ELF5 mRNAs. After blocked with AG490,the relative expression abundance of CSN2 and CSN3 mRNAs were reduced significantly in the co-culture group. In addition,the expressions of CSN2 and CSN3 mRNAs were inhibited significantly when both AG1024 and AG490 were supplemented. Conclusion UCMSCs can mediate JAK2 / STAT5 signaling pathwayvia IGF-1,and increase the expressions of milk protein synthesis key genes in BMECs,thus promoteing the synthesis of milk protein.
引文
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[20]Wang M Z,Ji Y,Wang C,et al.The preliminary study on the effects of growth hormone and insulin-like growth factor-Ⅰonκ-casein synthesis in bovine mammary epithelial cells in vitro[J].J Anim Physiol Anim Nutr(Berl),2016,100(2):251-255.
[21]Yang J X,Wang C H,Xu Q B,et al.Methionyl-methionine promotesα-s1 casein synthesis in bovine mammary gland explants by enhancing intracellular substrate availability and activating JAK2-STAT5 and mT OR-mediated signaling pathways[J].J Nutr,2015,145(8):1748-1753.
[22]黄田英,李庆章,侯晓明.乳腺中JAK/STAT信号通路的研究进展[J].中国乳品工业,2010,38(2):41-44.
[23]季昀,庞学燕,田青,等.生长激素和胰岛素样生长因子Ⅰ对奶牛乳蛋白合成关键激酶及调节因子mRNA表达量的影响[J].动物营养学报,2013,25(1):198-207.
[24]Szewczuk M.Association of a genetic marker at the bovine Janus kinase 2 locus(JAK2/RsaI)with milk production traits of four cattle breeds[J].J Dairy Res,2015,82(3):287-292.
[25]曹洋,艾阳,张源淑.乳蛋白合成的信号通路与营养调控[J].畜牧与兽医,2014,46(6):133-136.
[2]Gross J J,Bruckmaier R M.Repeatability of metabolic responses to a nutrient deficiency in early and midlactation and implications for robustness of dairy cows[J].J Dairy Sci,2015,98(12):8634-8643.
[3]Ha W T,Jeong H Y,Lee S Y,et al.Effects of the insulin-like growth factor pathway on the regulation of mammary gland development[J].Dev Reprod,2016,20(3):179-185.
[4]崔英俊,李庆章.奶牛不同发育时期乳腺组织IGF-Ⅰ及其受体的表达与定位[J].中国乳品工业,2014,42(6):4-6.
[5]邢媛媛,李大彪,李红磊,等.催乳素对奶牛乳腺上皮细胞乳脂和乳蛋白合成相关基因表达的影响[J].动物营养学报,2016,28(8):2439-2447.
[6]林忠荔,江明锋,任洪辉.牛乳蛋白合成的分子调控机制[J].中国畜牧兽医,2014,41(5):158-162.
[7]李文清,王加启,南雪梅,等.奶牛乳腺上皮细胞的不同培养方法比较及激素和细胞因子对β-酪蛋白mRNA表达的诱导[J].动物营养学报,2016,28(9):2607-2614.
[8]Lin J,Bao Z K,Zhang Q,et al.Transcriptome analysis of the mammary gland from GH transgenic goats during involution[J].Gene,2015,565(2):228-234.
[9]Gao H N,Hu H,Zheng N,et al.Leucine and histidine independently regulate milk protein synthesis in bovine mammary epithelial cells via mT OR signaling pathway[J].J Zhejiang Univ Sci B,2015,16(6):560-572.
[10]Shi H,Zhu J,Luo J,et al.Genes regulating lipid and protein metabolism are highly expressed inmammary gland of lactating dairy goats[J].Funct Integr Genomics,2015,15(3):309-321.
[11]Bian Y,Lei Y,Wang C,et al.Epigenetic regulation of miR-29s affects the lactation activity of dairy cow mammary epithelial cells[J].J Cell Physiol,2015,230(9):2152-2163.
[12]刘玲玲,赵龙,李晓丽,等.MSCs旁分泌对多柔比星诱导的H9C2细胞凋亡的影响[J].江苏大学学报(医学版),2014,24(3):221-225.
[13]赵艳坤.注射脐带间充质干细胞对荷斯坦公犊生长性能及血清生化指标的影响[D].新疆:新疆农业大学,2015.
[14]王立文,邵伟,赵艳坤,等.不同浓度奶牛乳腺上皮细胞和奶牛脐带间充质干细胞共培养对几种细胞因子分泌的影响研究[J].黑龙江畜牧兽医,2016,512(1):21-24.
[15]Laible G,Smolenski G,Wheeler T,et al.Increased gene dosage forβ-andκ-casein in transgenic cattle improves milk composition through complex effects[J/OL].Sci Rep,2016,23(6):37607.doi:10.1038/srep37607.
[16]刘正,刘亚兵,张进,等.乳及乳制品中蛋白质含量的测定[J].农业科技与信息,2014,432(7):24-25.
[17]徐连彬,林雪彦,胡志勇,等.氨基酸供给与内分泌互作调控乳蛋白合成的研究进展[J].动物营养学报,2016,28(8):2324-2333.
[18]Bao Z,Lin J,Ye L,et al.Modulation of mammary gland development and milk production by growth hormone expression in GH transgenic goats[J].Front Physiol,2016,29(7):278.doi:10.3389/fphys.2016.00278.eC ollection 2016.
[19]张童非.奶牛乳腺上皮细胞胰岛素样生长因子及受体IGF-ⅠR的表达与作用[D].哈尔滨:东北农业大学,2012.
[20]Wang M Z,Ji Y,Wang C,et al.The preliminary study on the effects of growth hormone and insulin-like growth factor-Ⅰonκ-casein synthesis in bovine mammary epithelial cells in vitro[J].J Anim Physiol Anim Nutr(Berl),2016,100(2):251-255.
[21]Yang J X,Wang C H,Xu Q B,et al.Methionyl-methionine promotesα-s1 casein synthesis in bovine mammary gland explants by enhancing intracellular substrate availability and activating JAK2-STAT5 and mT OR-mediated signaling pathways[J].J Nutr,2015,145(8):1748-1753.
[22]黄田英,李庆章,侯晓明.乳腺中JAK/STAT信号通路的研究进展[J].中国乳品工业,2010,38(2):41-44.
[23]季昀,庞学燕,田青,等.生长激素和胰岛素样生长因子Ⅰ对奶牛乳蛋白合成关键激酶及调节因子mRNA表达量的影响[J].动物营养学报,2013,25(1):198-207.
[24]Szewczuk M.Association of a genetic marker at the bovine Janus kinase 2 locus(JAK2/RsaI)with milk production traits of four cattle breeds[J].J Dairy Res,2015,82(3):287-292.
[25]曹洋,艾阳,张源淑.乳蛋白合成的信号通路与营养调控[J].畜牧与兽医,2014,46(6):133-136.