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
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
Vectors of pcDNA3.1-hTERT and pcDNA 3. 1-SV40 T were established. After linearization, they were cotransfected to mammary epithelial cells of Holstein cow, in order to research on the role of hTERT and SV40 T in immor...Vectors of pcDNA3.1-hTERT and pcDNA 3. 1-SV40 T were established. After linearization, they were cotransfected to mammary epithelial cells of Holstein cow, in order to research on the role of hTERT and SV40 T in immortalized mammary epithelial cells in vitro. Both PT-PCR and immunohistochemical as- says of cells were carried out. Results showed that the expression of hTERT and SV40 T could effectively prolong the culture time in vitro of mammary epithelial cells, and enhance the cell passage number. The obtained cell line could be expressed normally, indicating that the in vitro cultured mammary epithelial cells expressing both hTERT and SV40 T could effectively prolong cell llfe without affecting the characteristics of mammary cells.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
A galactopoietic compound, identified as dibutyl phthalate（DBP）, was isolated from Vaccaria segetalis. The activity of DBP on lactation ability of dairy cow mammary gland epithelial cells（DCMECs） cultured in vitro...A galactopoietic compound, identified as dibutyl phthalate（DBP）, was isolated from Vaccaria segetalis. The activity of DBP on lactation ability of dairy cow mammary gland epithelial cells（DCMECs） cultured in vitro and dairy cow was evaluated. Results showed that DBP could promote cell viability, proliferation ability, lactose and β-casein secretion of DCMECs, which could also raise the milk yields of dairy cows significantly.展开更多
To analyze miR-139 target sites in 3＇ UTR of GHR gene in dairy cow mammary gland, a GHR 3＇ UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy cow mammary g...To analyze miR-139 target sites in 3＇ UTR of GHR gene in dairy cow mammary gland, a GHR 3＇ UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy cow mammary gland epithelial cells （DCMECs）. The miR-139 targeting GHR 3＇ UTR was predicted by Target Scan 5.1 software, 3＇ UTR fragment of GHR was amplified by PCR from RNA of DCMECs. PCR products were cloned into Spe Ⅰ/Hind Ⅱ modified pMIR-Report vector. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into DCMECs using lipofectamine 2000 transfection reagent. The dualluciferase reporter assay system was used to quantitiate the reporter activity. The results showed that a 107 bp 3＇ UTR fragment of GHR gene was successfully cloned into the pMIR-Report vector, which authenticated by Spe Ⅰ/Hind Ⅲ digestion and DNA sequencing. The luciferase activity of reporter construction treated with miR-139 decreased 20.87% compared with the control group. It was concluded that the GHR3＇ UTR-luciferase reporter vector had been successfully constructed. The luciferase activity of the reporter could be suppressed by miR- 139.展开更多
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 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 Overseas Distinguished Experts Fund for Taishan Scholarthe Special Project for National Cow Industry Technology System Construction+3 种基金the Major Projects for National Transgene(2009ZX08007-006B,2011ZX08007-002,2011ZX08008-004)the Natural Science Foundation of Shandong Province(ZR2010CM012)the Innovation Projects for Jinan Universities and Institutes(201004027,201202059,201102034)the Youth Natural Science Foundation of Shandong Province(ZR2010ZR029)
文摘Vectors of pcDNA3.1-hTERT and pcDNA 3. 1-SV40 T were established. After linearization, they were cotransfected to mammary epithelial cells of Holstein cow, in order to research on the role of hTERT and SV40 T in immortalized mammary epithelial cells in vitro. Both PT-PCR and immunohistochemical as- says of cells were carried out. Results showed that the expression of hTERT and SV40 T could effectively prolong the culture time in vitro of mammary epithelial cells, and enhance the cell passage number. The obtained cell line could be expressed normally, indicating that the in vitro cultured mammary epithelial cells expressing both hTERT and SV40 T could effectively prolong cell llfe without affecting the characteristics of mammary cells.
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 "863" Project of Ministry of Science and Technology of China(2013AA102504-03)Talents Foundation of Northeast Agricultural University(2010RCB47,2010RCB55)
文摘A galactopoietic compound, identified as dibutyl phthalate（DBP）, was isolated from Vaccaria segetalis. The activity of DBP on lactation ability of dairy cow mammary gland epithelial cells（DCMECs） cultured in vitro and dairy cow was evaluated. Results showed that DBP could promote cell viability, proliferation ability, lactose and β-casein secretion of DCMECs, which could also raise the milk yields of dairy cows significantly.
基金Supported by the National Key Basic Research and Development (973) Plan (2011CB100804)Northeast Agricultural University Innovation Team Project (CXT005-1-1/CXT005-1-2)
文摘To analyze miR-139 target sites in 3＇ UTR of GHR gene in dairy cow mammary gland, a GHR 3＇ UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy cow mammary gland epithelial cells （DCMECs）. The miR-139 targeting GHR 3＇ UTR was predicted by Target Scan 5.1 software, 3＇ UTR fragment of GHR was amplified by PCR from RNA of DCMECs. PCR products were cloned into Spe Ⅰ/Hind Ⅱ modified pMIR-Report vector. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into DCMECs using lipofectamine 2000 transfection reagent. The dualluciferase reporter assay system was used to quantitiate the reporter activity. The results showed that a 107 bp 3＇ UTR fragment of GHR gene was successfully cloned into the pMIR-Report vector, which authenticated by Spe Ⅰ/Hind Ⅲ digestion and DNA sequencing. The luciferase activity of reporter construction treated with miR-139 decreased 20.87% compared with the control group. It was concluded that the GHR3＇ UTR-luciferase reporter vector had been successfully constructed. The luciferase activity of the reporter could be suppressed by miR- 139.
