The galactopoietic mechanism of Vaccaria segetalis is still unknown. Understanding dibutyl phthalate （DBP） separated from Vaccaria segetalis on the expression of lactation signal transduction genes of mammary gland ...The galactopoietic mechanism of Vaccaria segetalis is still unknown. Understanding dibutyl phthalate （DBP） separated from Vaccaria segetalis on the expression of lactation signal transduction genes of mammary gland epithelial cells, including prlr, erα, akt1, socs2, pparγ and elf5, will be helpful to reveal the molecular mechanism. Western blot and qRT- PCR were used to study the change of prlr, erα, akt, socs2, pparγ, and elf5 expression at mRNA and protein level. Co- localization expression of prolactin receptor （PRLR） and estrogen receptor α （ERα） was observed by immunofluorescence; the expression changes of miRNAs （21, 125b, 143, and 195） and the secretion of β-casein and lactose were detected by qRT-PCR and RP-HPLC. The results showed that Vaccaria segetalis active compound had similar fuctions as estrogen and/or prolactin （PRL） in dairy cow mammary gland epithelial cells （DCMECs）, increased the expressions of prlr, erα, akt1, and elf5 genes, while repressed pparγ expressions. DBP promoted socs2 mRNA expression, but its protein expressions were repressed. Furthermore, both DBP and PRL could repress the expressions of miRNA-125b, miRNA-143 and miRNA- 195 in DCMECs. DBP could repress the expression of miRNA-21, while the influence of PRL on miRNA-21 was not certain. DBP could promote the lactation ability of DCMECs by regulating the ER and PRLR cellular signal transduction pathway.展开更多
In previous experiment, we isolated a compound dibutyl phthalate （DBP） from Vaccaria segetalis which had galactopoietic function on mammary gland epithelial cells of dairy cow （DCMECs）. In this experiment, we asce...In previous experiment, we isolated a compound dibutyl phthalate （DBP） from Vaccaria segetalis which had galactopoietic function on mammary gland epithelial cells of dairy cow （DCMECs）. In this experiment, we ascertained the metabolic regulation function of DBP on DCMECs. Many genes related to lactation including Stat5, AMPK, b-casein, Glut1, SREBP-1, PEPCK, and ACC were detected by real-time PCR. Furthermore, Stat5 and AMPK were detected by Western blot and immunofluorescence co-localization, respectively. The results showed that DBP stimulates the expression of Stat5 and p-Stat5, thus activates Stat5 cell signal transduction pathway and stimulates b-casein synthesis. DBP also raises the activities of Glut1 and AMPK to stimulate glucose uptake and glycometabolism and activates the expression of AMPK downstream target genes PEPCK and ACC and expression of SREBP-1 to stimulate milk fat synthesis. In addition, the activities of HK, G-6-PDH, ICDH, ATPase, and energy charges were stimulated by DBP to increase the energy metabolism level of DCMECs. The results showed DBP stimulates energy metabolism related to galactopoietic function in DCMECs.展开更多
To investigate the expression of antibacterial peptide CecropinB cDNA in dairy goat mammary gland epithelial cells,the CecropinB gene was cloned and was inserted into a eukaryotic vector pECFP-C1 to construct the reco...To investigate the expression of antibacterial peptide CecropinB cDNA in dairy goat mammary gland epithelial cells,the CecropinB gene was cloned and was inserted into a eukaryotic vector pECFP-C1 to construct the recombinant plasmid pECFP-B by genetic engineering technique.Recombinant plasmid pECFP-B was transfected into dairy goat mammary gland epithelial to detect the bactericidal activity of CecropinB.The expression of CecropinB was also detected.The result of RT-PCR demonstrated CecropinB gene was expressed in transfected cells.CecropinB recombinant plasmid DNA was injected into udders and CecropinB was expressed in mammary gland,exhibiting bactericidal activity to Staphylococcus aureus in vivo experiments.展开更多
Mammary epithelial cells with lactational function can be a valuable cellular model for research of the development and regulation of the mammary gland.This paper describes some aspects of function of an epithelial ce...Mammary epithelial cells with lactational function can be a valuable cellular model for research of the development and regulation of the mammary gland.This paper describes some aspects of function of an epithelial cell line from the mammary gland of the dairy goat.SDS-PAGE,triglyceride and lactose content of cultured cells were used to assess synthetic function of cells and the effects of exposure to insulin and prolactin.Results show that goat mammary epithelial cells can synthesize fat,proteins and lactose when they were cultured in DMEM-F12 medium with added EGF,IGF-1,ITS and FBS.There were no obvious changes after 48h treatment with additional insulin.Prolactin added to the basal medium significantly increased synthesis of proteins and lactose.A mammary gland epithelial cell line from goats which has lactational function has been established.This outcome provides a valuable and convenient model system.展开更多
The paper was to study the effects of different concentrations of lipopelysaccharide （LPS） on expression of nuclear factor Kappa B （NF-κB） and lingual antimicrobial peptide （LAP） gene in mammary epithelial ceil...The paper was to study the effects of different concentrations of lipopelysaccharide （LPS） on expression of nuclear factor Kappa B （NF-κB） and lingual antimicrobial peptide （LAP） gene in mammary epithelial ceils of dairy cow. The mammary epithelial ceils of dairy cow were stimulated by different concentrations （50, 100,200,400 and 800 ng/mL） of LPS. The total RNA of cells was extracted after stimulation for 2, 4, 8, 16, 24, 48 and 72 h, respectively, and the mRNA expression levels of NF-κB P65 and LAP were evaluated by real-time quantitative PCR. The results showed that the expression of NF-κB P65 and LAP mRNA treated with 400 ng/mL LPS for 72 h were the highest compared to the control group （ P 〈0.01 ）. The result confirmed that the expression activity of NF-κB was enhanced in inflammatory effects of inammary epithelial cells induced by LPS, which regulated the expression of defense gene LAP, with certain dose and time effects.展开更多
[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer techn...[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.展开更多
基金supported by the National High Tech-nologies R&D Program (863 Program) of China(2006AA10Z1A4)the Innovation Team Project of Northeast Agricultural University, China (LXT005-1-2)
文摘The galactopoietic mechanism of Vaccaria segetalis is still unknown. Understanding dibutyl phthalate （DBP） separated from Vaccaria segetalis on the expression of lactation signal transduction genes of mammary gland epithelial cells, including prlr, erα, akt1, socs2, pparγ and elf5, will be helpful to reveal the molecular mechanism. Western blot and qRT- PCR were used to study the change of prlr, erα, akt, socs2, pparγ, and elf5 expression at mRNA and protein level. Co- localization expression of prolactin receptor （PRLR） and estrogen receptor α （ERα） was observed by immunofluorescence; the expression changes of miRNAs （21, 125b, 143, and 195） and the secretion of β-casein and lactose were detected by qRT-PCR and RP-HPLC. The results showed that Vaccaria segetalis active compound had similar fuctions as estrogen and/or prolactin （PRL） in dairy cow mammary gland epithelial cells （DCMECs）, increased the expressions of prlr, erα, akt1, and elf5 genes, while repressed pparγ expressions. DBP promoted socs2 mRNA expression, but its protein expressions were repressed. Furthermore, both DBP and PRL could repress the expressions of miRNA-125b, miRNA-143 and miRNA- 195 in DCMECs. DBP could repress the expression of miRNA-21, while the influence of PRL on miRNA-21 was not certain. DBP could promote the lactation ability of DCMECs by regulating the ER and PRLR cellular signal transduction pathway.
基金supported by the National High Technology Research and Development Program of China (863 Program, 2006AA10Z1A4)the Innovation Team of the Northeast Agricultural University, China (LXT005-1-2)the Talents Foundation of Northeast Agriculture Univesity, China (2010RCB47)
文摘In previous experiment, we isolated a compound dibutyl phthalate （DBP） from Vaccaria segetalis which had galactopoietic function on mammary gland epithelial cells of dairy cow （DCMECs）. In this experiment, we ascertained the metabolic regulation function of DBP on DCMECs. Many genes related to lactation including Stat5, AMPK, b-casein, Glut1, SREBP-1, PEPCK, and ACC were detected by real-time PCR. Furthermore, Stat5 and AMPK were detected by Western blot and immunofluorescence co-localization, respectively. The results showed that DBP stimulates the expression of Stat5 and p-Stat5, thus activates Stat5 cell signal transduction pathway and stimulates b-casein synthesis. DBP also raises the activities of Glut1 and AMPK to stimulate glucose uptake and glycometabolism and activates the expression of AMPK downstream target genes PEPCK and ACC and expression of SREBP-1 to stimulate milk fat synthesis. In addition, the activities of HK, G-6-PDH, ICDH, ATPase, and energy charges were stimulated by DBP to increase the energy metabolism level of DCMECs. The results showed DBP stimulates energy metabolism related to galactopoietic function in DCMECs.
基金Supported by Key Teachers Foundation of Education Office of Heilongjiang Province,2005(1055G005)
文摘To investigate the expression of antibacterial peptide CecropinB cDNA in dairy goat mammary gland epithelial cells,the CecropinB gene was cloned and was inserted into a eukaryotic vector pECFP-C1 to construct the recombinant plasmid pECFP-B by genetic engineering technique.Recombinant plasmid pECFP-B was transfected into dairy goat mammary gland epithelial to detect the bactericidal activity of CecropinB.The expression of CecropinB was also detected.The result of RT-PCR demonstrated CecropinB gene was expressed in transfected cells.CecropinB recombinant plasmid DNA was injected into udders and CecropinB was expressed in mammary gland,exhibiting bactericidal activity to Staphylococcus aureus in vivo experiments.
基金supported by Innovation Team Project of Northeast Agricultural Vniversity(XLT005-1-2)Research Fund for the Doctoral Program of Heilongjiang Educational Committee(HLJBSDJI2004-15)
文摘Mammary epithelial cells with lactational function can be a valuable cellular model for research of the development and regulation of the mammary gland.This paper describes some aspects of function of an epithelial cell line from the mammary gland of the dairy goat.SDS-PAGE,triglyceride and lactose content of cultured cells were used to assess synthetic function of cells and the effects of exposure to insulin and prolactin.Results show that goat mammary epithelial cells can synthesize fat,proteins and lactose when they were cultured in DMEM-F12 medium with added EGF,IGF-1,ITS and FBS.There were no obvious changes after 48h treatment with additional insulin.Prolactin added to the basal medium significantly increased synthesis of proteins and lactose.A mammary gland epithelial cell line from goats which has lactational function has been established.This outcome provides a valuable and convenient model system.
基金Supported by National Natural Science Foundation of China(30460093,31060328)Projects for Key Areas of Scientific Research in Inner Mongolia Colleges and Universities(ZL010144)Major Projects for Cultivation of New Breeds of Genetically Modified Organisms(2008ZX08007-004)
文摘The paper was to study the effects of different concentrations of lipopelysaccharide （LPS） on expression of nuclear factor Kappa B （NF-κB） and lingual antimicrobial peptide （LAP） gene in mammary epithelial ceils of dairy cow. The mammary epithelial ceils of dairy cow were stimulated by different concentrations （50, 100,200,400 and 800 ng/mL） of LPS. The total RNA of cells was extracted after stimulation for 2, 4, 8, 16, 24, 48 and 72 h, respectively, and the mRNA expression levels of NF-κB P65 and LAP were evaluated by real-time quantitative PCR. The results showed that the expression of NF-κB P65 and LAP mRNA treated with 400 ng/mL LPS for 72 h were the highest compared to the control group （ P 〈0.01 ）. The result confirmed that the expression activity of NF-κB was enhanced in inflammatory effects of inammary epithelial cells induced by LPS, which regulated the expression of defense gene LAP, with certain dose and time effects.
基金Supported by the National High-tech R&D Program(2004AA213072)the Doctor Fund of Henan University of Science and Technology~~
文摘[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.
