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1.
ZHANG KezhongJIANG PeihongLU DaruHUANG WeidaCHEN LiXUE Jerry L. HsuehQIU Xinfang 《中国科学:生命科学英文版》1998,41(4):406-412
Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β-casein
gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were transferred to lactating mice via tail
vein. Both hFIX and Lac2 gene could be expressed in the mammary gland of the treated mice. The highest production of hFIX
protein was 80.28 ng per mL milk, and more than 85% of hFIX protein appeared to be γ-carboxylation and biologically active.
The results suggested that the 2.0 kb sequence of β-casein gene including promoter, exon 1 was effective to drive hFIX gene
expression in mammary gland and intron 1 of β-casein gene had an effect on the tissue specific expression. The expression
level in mouse milk injected with hFIX minigene vector containing hFIX endogenous intron 1 was increased by above 3 times
of that injected with hFIX cDNA vector.
Project supported by the State High Technology Development Program and Shanghai Science and Technology Commission. 相似文献
2.
J. E. Keys A. J. Guidry E. Cifrian 《In vitro cellular & developmental biology. Animal》1997,33(3):201-205
Summary A flow cytometric technique was developed to measure the relative concentration of whey protein and β-casein in individual
fixed and permeabilized bovine mammary epithelial cells. Primary bovine mammary epithelial cells were compared to mammary
cells isolated from explants after a 24-h incubation and a bovine mammary epithelial transfected cell line (MAC-T). Cells
were incubated with rabbit anti-bovine whey protein (α-lactalbumin + β-lactoglobulin) or β-casein primary antibodies followed
by a fluorescein-labeled goat anti-rabbit IgG second antibody. The number and intensity of fluorescing cells were measured
using an EPICS Profile Flow Cytometer. Primary and explant cells contained 3.3 and 2.8 times more whey protein than MAC-T
cells. Explant epithelial cells contained 2.9 and 5.1 times more β-casein than primary or MAC-T cells. The higher concentrations
of specific proteins within the cells was attributed to either greater synthesis or reduced secretion. These data show that
flow cytometry is capable of detecting differences in milk protein concentration in different mammary epithelial cell types. 相似文献
3.
Establishment and characterization of a lactating dairy goat mammary gland epithelial cell line 总被引:1,自引:0,他引:1
To study milk synthesis in dairy goat mammary gland, we had established an in vitro lactating dairy goat mammary epithelial
cell (DGMEC) line. Mammary tissues of Guan Zhong dairy goats at 35 d of lactation were dispersed and cultured in a medium
containing epithelial growth factor, insulin-like growth factor-1, insulin transferrin serum, and fetal bovine serum. Epithelial
cells were enriched by digesting with 0.25% trypsin repeatedly to remove fibroblast cells and were identified as epithelial
origin by staining with antibody against cytokeratine 18. The DGMECs displayed monolayer, cobble-stone, epithelial-like morphology,
and formed alveoli-like structures and island monolayer aggregates which were the typical characteristics of mammary epithelial
cells. A one-half logarithmically growth curve and cytoplasmic lipid droplets in these cells were observed. In this paper,
we also studied the lactating function of DGMECs. Results showed that DGMECs could secrete lactose and β-casein. Lactating
function of the cells had no obvious change after 48 h treated by insulin, while prolactin could obviously raise the secretion
of milk proteins and lactose. 相似文献
4.
5.
Heregulin-α (HRGα) is a cytokine secreted by the mammary mesenchyme, adjacent to lobuloalveolar structures. To understand
the role of HRGα and its receptors in mammary glands, and the underlying mechanisms, we performed this study to determine
the expression and localization of HRGα and its receptors ErbB2 and ErbB3. We also determined the role of HRGα in the development
of mammary glands, β-casein expression and secretion, Rab3A protein expression and the phosphorylation of HRGα signaling molecules
using confocal laser scanning microscopy, tissue culture, capillary electrophoresis, Western blotting and enzyme-linked immunosorbent
assays. We found that a peak was on pregnancy day 15. Changes of ErbB2 and ErbB3 expression were positively and linearly correlated
with HRGα, indicating that HRGα positively regulates ErbB2 and ErbB3 expression. During pregnancy, HRGα enhanced the phosphorylation
of STAT5, p42/p44, p38, PKC and Rab3A protein expression, stimulated the proliferation and differentiation of the ductal epithelial
cells of mammary glands, and increased and maintained the expression and secretion of β-casein. During lactation, HRGα enhanced
the phosphorylation of STAT5 and p38, inhibited the phosphorylation of PKC and Rab3A protein expression, maintained the morphology
of the mammary glands and increased the secretion of lactoprotein to reduce the expression of β-casein in mammary epithelial
cells. During involution, HRGα induced the phosphorylation of STAT3 and Rab3A protein expression, and inhibited the phosphorylation
of PKC to stimulate the degeneration of mammary epithelial cells. It also inhibited the secretion of β-casein, resulting in
increased levels of β-casein in mammary epithelial cells. 相似文献
6.
Marcelo G. Cerdn Juan I. Young Esilda Zino Toms L. Falzone Vernica Otero Hctor N. Torres Marcelo Rubinstein 《Molecular reproduction and development》1998,49(3):236-245
The spatial, temporal, and hormonal pattern of expression of the β-casein gene is highly regulated and confined to the epithelial cells of the lactating mammary gland. Previous studies have shown that 1.7 kb of the bovine β-casein promoter were able to drive cell-specific and hormone-dependent expression to a mouse mammary cell line but failed to induce accurate expression to the mammary gland of transgenic mice. We investigated here the ability of 3.8 kb of the bovine β-casein gene promoter to drive the expression of the human growth hormone (hGH) gene in transgenic mice. A Northern blot analysis using total RNA obtained from different tissues of lactating and nonlactating females revealed the presence of hGH mRNA only in the mammary gland of lactating females. hGH mRNA was not detectable in the mammary gland of virgin females or males. A developmental analysis showed that hGH mRNA only peaked on parturition, resembling more closely the bovine β-casein temporal expression pattern rather than the murine. In situ hibridization studies performed on mammary gland sections showed that the cellular pattern of hGH expression was homogeneous in all lobules from heterozygous and homozygous transgenic mice. Silver grain counts on the tissue sections highly correlated with the hGH contents in the milk determined by radioimmunoassay (r = 0.996). Thus 3.8 kb of the bovine β-casein promoter direct a high-level expression of a reporter gene to the lactating mammary gland of transgenic mice in a tissue-specific and developmentally regulated manner. Mol. Reprod. Dev. 49:236–245, 1998. © 1998 Wiley-Liss, Inc. 相似文献
7.
Little is known about the transport of iron into the mammary secretory cell and the process of milk iron secretion. The concentration of iron in milk is remarkably unaffected by maternal iron status, suggesting that the uptake of iron into the mammary gland is regulated. It is known that iron enters other cells via transferrin receptor-mediated endocytosis. This study was designed to isolate and characterize the mammary gland transferrin receptor in lactating rat mammary tissue using immunochemical techniques. The existence of functional mammary gland transferrin receptors in lactating rodents was demonstrated using radiolabel-binding techniques. Isolation of mammary transferrin receptors by affinity chromatography was confirmed using immunoelectrophoresis and slot blot analysis. The intact transferrin receptor was found to have a molecular weight of 176 kd as determined by Western blotting followed by scanning densitometry. Reduction of the receptor with beta-mercaptoethanol gave a molecular weight of 98 kd. An additional immunoreactive band of 135 kd was observed. The presence of transferrin receptors in normal lactating rat mammary tissue is likely to explain iron transport into mammary tissue for both cellular metabolism and milk iron secretion. 相似文献
8.
Alain Pauloin Sophie Chat Christine Péchoux Catherine Hue-Beauvais Stéphanie Droineau Laurent Galio Eve Devinoy Eric Chanat 《Cell and tissue research》2010,340(1):91-102
Although virtually all cells store neutral lipids as cytoplasmic lipid droplets, mammary epithelial cells have developed a
specialized function to secrete them as milk fat globules. We have used the mammary epithelial cell line HC11 to evaluate
the potential connections between the lipid and protein synthetic pathways. We show that unsaturated fatty acids induce a
pronounced proliferation of cytoplasmic lipid droplets and stimulate the synthesis of adipose differentiation-related protein.
Unexpectedly, the cellular level of β-casein, accumulated under lactogenic hormone treatment, decreases following treatment
of the cells with unsaturated fatty acids. In contrast, saturated fatty acids have no significant effect on either cytoplasmic
lipid droplet proliferation or cellular β-casein levels. We demonstrate that the action of unsaturated fatty acids on the
level of β-casein is post-translational and requires protein synthesis. We have also observed that proteasome inhibitors potentiate
β-casein degradation, indicating that proteasomal activity can destroy some cytosolic protein(s) involved in the process that
negatively controls β-casein levels. Finally, lysosome inhibitors block the effect of unsaturated fatty acids on the cellular
level of β-casein. Our data thus suggest that the degradation of β-casein occurs via the microautophagic pathway. 相似文献
9.
Ornithine decarboxylase (ODC), antizyme (AZ), and antizyme inhibitor (AIn) play a key role in regulation of intracellular
polyamine levels by forming a regulatory circuit through their interactions. To gain insight into their functional importance
in cell growth and differentiation, we systematically examined the changes of their expression, cellular polyamine contents,
expression of genes related to polyamine metabolism, and β-casein gene expression during murine mammary gland development.
The activity of ODC and AZ1 as well as putrescine level were low in the virgin and involuting stages, but they increased markedly
during late pregnancy and early lactation when mammary cells proliferate extensively and begin to augment their differentiated
function. The level of spermidine and expression of genes encoding spermidine synthase and AIn increased in a closely parallel
manner with that of casein gene expression during pregnancy and lactation. On the other hand, the level of spermidine/spermine
N
1-acetyltransferase (SSAT) mRNA and AZ2 mRNA decreased during those periods. Immunohistochemical analysis showed the translocation
of ODC and AIn between the nucleus and cytoplasm and the continuous presence of AZ in the nucleus during gland development.
Reduction of AIn by RNA interference inhibited expression of β-casein gene stimulated by lactogenic hormones in HC11 cells.
In contrast, reduction of AZ by AZsiRNA resulted in the small increase of β-casein gene expression. These results suggested
that AIn plays an important role in the mammary gland development by changing its expression, subcellular localization, and
functional interplay with AZ. 相似文献
10.
Gordon Parry Debble Farson Betsey Cullen Mina J. Bissell 《In vitro cellular & developmental biology. Plant》1988,24(12):1217-1222
Summary Primary cultures of mouse mammary epithelial cells synthesize significant quantities of chondroitin and heparan sulfate proteoglycans
(16). Long term treatment of such cultures with p-nitrophenyl-β-D-xylopyranoside leads to a 10–20 fold increase in the synthesis
and secretion of free chondroitin sulfate glycosaminoglycan (GAG) chains and assembly of a cell-associated matrix that is
relatively enriched in heparan sulfate proteoglycan.
This modulation of cell-synthesized proteoglycans leads to significant changes in cell morphology and cellular differentiation.
Notably cells cultured on plastic culture dishes change from being flattened to cuboidal. The synthesis of the milk proteins
α1, α2, and β-casein is also increases as is the formation of fat droplets and fat droplet membrane components. Promotion
of differentiation increases with increasing xyloside concentration in the range 0–1.5 mM, but there may be a block in secretion
at higher xyloside concentrations.
While the detailed mechanisms remain to be elucidated, we conclude that the composition of proteoglycans incorporated into
the matrix (and possibly the glycosaminoglycans secreted into the medium), may play a significant role in maintaining the
phenotypic characteristics of terminally differentiated mammary epithelial cells.
This research was supported by the Office of Health and Environmental Research, Office of Energy Research, U.S. Dept. of Energy
under contract No. DEAC-03-76SF00098 and by National Institutes of Health Grant CA44398-01 (G. Parry)
Editor's Statement Exogenous elements of extracellular matrix affect expression of cultured mammary cell function. This work
reports manipulation of cell-derived endogenous matrix elements and shows correlative changes in cell functions. 相似文献
11.
Ariela Baruch Moshe Shani David R. Hurwitz Itamar Barash 《Genesis (New York, N.Y. : 2000)》1995,16(3):241-252
12.
13.
Pten作为抑癌基因,参与调控细胞生长、粘附、凋亡以及其它细胞活动.目前,国内外关于Pten在奶牛乳腺发育过程中表达及调节的研究鲜有报道.为了揭示Pten的表达与奶牛乳腺发育与泌乳之间的关系,本研究应用qRT-PCR技术检测Pten在不同泌乳时期和不同乳品质的奶牛乳腺组织中的表达差异,进而应用脂质体转染方法,通过siRNA介导的RNA干扰技术改变Pten基因在奶牛乳腺上皮细胞中的表达量,CASY法检测细胞活力,用ELISA试剂盒检测细胞分泌β-酪蛋白的含量,采用qRT-PCR、Western 印迹等技术检测Pten对奶牛乳腺上皮细胞中乳蛋白相关信号通路基因表达的影响.结果显示,泌乳期高乳品质奶牛乳腺组织中Pten表达水平显著低于泌乳期低乳品质及干乳期奶牛;Pten基因沉寂后,细胞活力提高,β-酪蛋白质量浓度增加,CSN2、AKT、MTOR、STAT5表达量增加.研究表明,Pten可通过抑制细胞活力和乳蛋白分泌而影响泌乳. 相似文献
14.
Leptin is an autocrine and paracrine factor which affects the development of duct, formation of gland alveolus, expression
of milk protein gene and onset involution of mammary gland. In order to know the function and mechanism of leptin in mammary
gland, the protein expression and localization of leptin and its long form receptor (OB-Rb) were detected by a confocal laser
scanning microscope. To study the impacts of leptin on mammary gland and leptin signal transduction pathway in pregnancy-,
lactation-and involution-stage mammary gland, explants were cultured and Western blotting was used. The results showed that
in the whole development cycle of mammary gland, the expression of leptin and OB-Rb was in positive correlation. In virgin
the leptin expression was the highest and then decreased in pregnancy. In lactation the expression of leptin was low and upgraded
in involution, and recovered to the original level about virgin on involution 13 d. The localization of leptin and OB-Rb revealed
that leptin induced the expression of OB-Rb specifically and controlled the development and physiological function of the
mammary gland by binding to OB-Rb. In pregnancy stage, leptin stimulated proliferation and differentiation of ductal epithelial
cells by JAK-MAPK signal pathway. In lactation, leptin induced gene expression of β-casein by JAK-STAT5 signal pathway, and
in involution leptin induced mammary epithelial cell apoptosis and mammary gland restitution by JAK-STAT3 signal pathway. 相似文献
15.
Kwon DN Song H Park JY Lee SY Cho SK Kang SJ Jang JS Seo HG Kim JH 《Transgenic research》2006,15(1):37-55
We analyzed two transgenic mouse lines that secrete rhEPO in their milk to assess the dynamic control of N-linked oligosaccharides.
Since pharmaceutically available epoetin α and β are produced in CHO cells, we compared transgenic mammary gland-derived rhEPO
to its CHO cell-derived counterpart. The major glycosyltransferases that determine the N-oligosaccharides patterns of rhEPO
include N-acetylglycosaminyltransferase (GnT) and α1,3/4 fucosyltransferase (Fuc-TIV), GnT-III, -V and Fuc-TIV expression
in the mouse mammary gland is significantly higher than that in Chinese hamster ovary (CHO)-derived cells, where the protein
is not detectable. The data suggest that N-linked sugar chain patterns of recombinant glycoproteins, produced by the mammary
gland differ, since GnT-III alters the sugar pattern extensively. In our experiments, rhEPO produced by the transgenic mice
contains more tetra-acidic oligosaccharide structures than epoetin α derived from CHO cells, a rhEPO that is widely used therapeutically.
Accordingly, we examined milk-derived rhEPO activity, both in vitro and in vivo. The rhEPO protein purified from the milk of mammary glands upregulates the EPO receptor-mediated expression of the STAT5
gene in MCF-7 cells in a dose-dependent manner, similar to the effects of epoetin α. Furthermore, direct injection of rhEPO
into the mouse tail vein leads to an increase in the levels of blood components, such as red blood cells and platelets. In
light of these findings, we suggest that the mammary glands of transgenic animals provide a sufficient environment to generate
rhEPO with post-translational modifications for biopharmaceutical use.
These authors are equal contributors to this work. 相似文献
16.
Leptin is an autocrine and paracrine factor which affects the development of duct, formation of gland alveolus, expression of milk protein gene and onset involution of mammary gland. In order to know the function and mechanism of leptin in mammary gland, the protein expression and localization of leptin and its long form receptor (OB-Rb) were detected by a confocal laser scanning microscope. To study the impacts of leptin on mammary gland and leptin signal transduction pathway in pregnancy-, lacta-tion-and involution-stage mammary gland, explants were cultured and Western blotting was used. The results showed that in the whole development cycle of mammary gland, the expression of leptin and OB-Rb was in positive correlation. In virgin the leptin expression was the highest and then decreased in pregnancy. In lactation the expression of leptin was low and upgraded in involution, and recovered to the original level about virgin on involution 13 d. The localization of leptin and OB-Rb revealed that leptin induced the expression of OB-Rb specifically and controlled the development and physiological function of the mammary gland by binding to OB-Rb. In pregnancy stage, leptin stimulated proliferation and differentiation of ductal epithelial cells by JAK-MAPK signal pathway. In lactation, leptin induced gene expression of β-casein by JAK-STAT5 signal pathway, and in involution leptin induced mammary epithelial cell apoptosis and mammary gland restitution by JAK-STAT3 signal pathway. 相似文献
17.
Cardiac fatty acid binding protein (cFABP) is abundantly expressed in the nondividing, functionally differentiated mammary ephithelium. It is very closely related, if not identical to, a previously described protein termed mammary derived growth inhibitor (MDGI). In vitro studies suggest that low concentrations of diffusible cFABP/MDGI may play a hormone-like role in limiting proliferative activity and promoting functional differentiation of this tissue, but no in vivo data to support this idea have been published. To test this hypothesis, we compared the levels of cFABP mRNA with both the epithelial DNA labelling index and levels of β-casein mRNA in wild-type mice. We also investigated the effect of a precocious experimental increase of cFABP levels in the mammary gland of transgenic mice on the labelling index and β-casein mRNA levels. This was accomplished by expressing a bovine cFABP cDNA under the control of the ovine β-lactoglobulin (BLG) gene promoter. We found that although both the DNA labelling index, β-casein mRNA levels, and cFABP mRNA levels in wild-type mice are developmentally regulated, they do not correlate with each other during early pregnancy in individual mice. Moreover, a three- to fourfold increase of total cFABP mRNA in two transgenic lines did not affect the DNA labelling index or the levels of β-casein mRNA, an established marker of differentiation of the mammary epithelium, at this developmental stage. These data suggest that epithelial DNA synthesis, β-casein gene expression, and expression of the cFABP gene are regulated independently in the proliferatively active mammary gland and that the rapidly dividing mammary epithelial cells are not susceptible to the action of cFABP during early pregnancy. © 1995 Wiley-Liss, Inc. 相似文献
18.
19.
The regulation of milk constituents, synthesis and secretion in tissue cultures of the bovine mammary gland was altered by a whey fraction of bovine milk. α-Casein gene expression, casein secretion and fatty acid synthesis were inhibited by the whey fraction in a dose-dependent manner. The whey fraction inhibited the enhancement activity of prolactin on α-casein gene expression and fatty acid synthesis, and also inhibited casein secretion to the medium, in explants cultured in a medium with or without prolactin. No effect on the expression of the β-lactoglobulin gene was found. 相似文献
20.
Milk Protein Synthesis, Gene Expression, and Hormonal Responsiveness in Primary Cultures of Mammary Cells from Lactating Sheep 总被引:1,自引:0,他引:1
Thomas T. Wheeler Megan R. Callaghan Stephen R. Davis Colin G. Prosser Richard J. Wilkins 《Experimental cell research》1995,217(2)
A ruminant mammary cell culture that accurately reproduces mammary function in vitro would be a valuable tool in studies of ruminant lactation, With this in mind, we have examined milk protein synthesis and secretion, milk protein mRNA abundance, and hormonal responsiveness in primary cultures of mammary acini from lecturing sheep. α- and β-casein protein synthesis, β-lactoglobulin synthesis, and α-casein, β-casein, and β-lactoglobulin secretion are maintained at high levels for 8 h in culture, but then decline to approximately 25% of maximal rates between 8 and 24 h in culture, whereas synthesis of other proteins remains unaltered. The relative abundance of α-S1-casein, β-lactoglobulin, and α-lactalbumin mRNAs similarly decline between 8 and 24 h in culture. Extracellular labeled α-casein is increased fourfold in the presence of fetal calf serum (FCS). In total, FCS alters the abundance of 47 of 68 secreted proteins detected by two-dimensional electrophoresis. However, FCS and lactogenic/galactopoietic hormones had no effect on the rate of decline of mammary function and did not promote any regaining of function when present for up to 9 days in culture. These results suggest that providing its limitations are recognized, this primary cell culture system may be useful in studying some aspects of ruminant mammary function in vitro. 相似文献