RESUMO
The aim of this study was to construct a mammary gland-specific expression vector, pGN, and to validate its function in expressing growth hormone (GH) both in vitro and in vivo. First, the GH gene was amplified and inserted downstream of the b-casein 5'-arm. Next, the neo gene was cloned downstream of the b-casein 3'-arm as a selection marker. To analyze the bioactivity of the pGN plasmid, we expressed pGN in a Bcap-37 cell line and in the goat mammary gland. Quantitative PCR analysis revealed that the expression of GH mRNA in the pGN-transfected group was higher than that of the control group in Bcap-37 cells. Results of a radioimmunoassay and an enzyme-linked immunosorbent assay demonstrated that the pGN-transfected group expressed much more GH protein than the non-transfected group (P < 0.05). Upon injection of the pGN plasmid into the goat mammary gland, GH mRNA and growth hormone receptor mRNA expressions increased 2-fold. In vivo analyses revealed that GH protein expression was higher in the injected group than in the control group. Together, these results strongly demonstrated that the pGN plasmid was constructed correctly and exhibited favorable bioactivity in efficiently expressing GH both in vitro and in vivo.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hormônio do Crescimento/genética , Glândulas Mamárias Animais/crescimento & desenvolvimento , Plasmídeos/genética , Animais , Caseínas/biossíntese , Caseínas/genética , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Cabras , Hormônio do Crescimento/biossíntese , Humanos , Glândulas Mamárias Animais/metabolismo , Especificidade de Órgãos , RNA Mensageiro/biossíntese , TransfecçãoRESUMO
The objective of this study was to characterize the genetic architecture underlying the absolute concentrations of 2 important milk proteins, κ-casein (κ-CN) and ß-lactoglobulin (ß-LG), in a backcross population of (Holstein × Jersey) × Holstein cattle. A genome-wide association analysis was performed using a selective DNA pooling strategy and the Illumina BovineHD BeadChip assay [777,000 (777K) SNP markers; Illumina Inc., San Diego, CA]. After correction for multiple testing, 25 single nucleotide polymorphisms were found to be associated with κ-CN and 36 single nucleotide polymorphisms were associated with ß-LG. A pathway association analysis revealed 15 Gene Ontology (GO) terms associated with the κ-CN trait and 28 GO terms associated with ß-LG. In addition, several GO terms were associated with both milk proteins. Further analysis revealed that κ-CN and ß-LG production is regulated by both kinase and phosphatase activity, including mechanisms regulating the extracellular matrix. These results are in concordance with the complex multihormonal process controlling the expression of milk proteins and interactions between mammary epithelial cells and extracellular matrix components. Although κ-CN and ß-LG milk proteins are expressed by single genes, the results from this study showed that many loci are involved in the regulation of the concentration of these 2 proteins.
Assuntos
Caseínas/genética , Bovinos/genética , Lactoglobulinas/genética , Animais , Caseínas/análise , Caseínas/biossíntese , Cromatografia Líquida de Alta Pressão/veterinária , Estudo de Associação Genômica Ampla/veterinária , Genótipo , Lactoglobulinas/análise , Lactoglobulinas/biossíntese , Leite/química , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Característica Quantitativa HerdávelRESUMO
Epithelial cells from mammary gland tissue that are cultured in vitro are able to maintain specific functions of this gland, such as cellular differentiation and milk protein synthesis. These characteristics make these cells a useful model to study mammary gland physiology, development and differentiation; they can also be used for production of exogenous proteins of pharmaceutical interest. Bovine mammary epithelial cells were cultured in vitro after isolation from mammary gland tissue of animals at different stages of development. The cells were plated on Petri dishes and isolated from fibroblasts using saline/EDTA treatment, followed by trypsinization. Cells isolated on plastic were capable of differentiating into alveolus-like structures; however, only cells derived from non-pregnant and non-lactating animals expressed ß-casein. Real-time qPCR and epifluorescence microscopy analyses revealed that alveolus-like structures were competent at expressing Emerald green fluorescent protein (EmGFP) driven by the ß-casein promoter, independent of ß-casein expression.
Assuntos
Caseínas/biossíntese , Caseínas/genética , Células Epiteliais/citologia , Glândulas Mamárias Animais/embriologia , Proteínas do Leite , Animais , Caseínas/metabolismo , Bovinos , Diferenciação Celular , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde , Lactação/fisiologia , Lentivirus/genética , Glândulas Mamárias Animais/citologia , Proteínas do Leite/biossíntese , Proteínas do Leite/genética , Proteínas do Leite/metabolismo , Reação em Cadeia da Polimerase , Gravidez , Regiões Promotoras GenéticasRESUMO
Mammary gland morphogenesis and differentiation are mediated through the combined activities of systemic hormones and locally synthesized growth factors. Activin, a member of the transforming growth factor (TGF)-beta superfamily, is known to regulate the growth and differentiation of several cell types. In the present study, we investigated the role of activin in rat mammary gland on different stages of development. We found that activin A in vitro inhibits the proliferation of isolated acini, and this effect increases with the development of the gland. This factor also produces in vitro an inhibition of the final differentiation of acini obtained from 19th day pregnant rats. We also report the expression of activin receptors IIA and IIB mRNA in whole rat mammary gland and acini, with decreased levels of expression of type IIA (in both compartments) and IIB (in acini) during pregnancy and lactogenesis. In addition, we show that activin betaB-subunit mRNA decreases throughout pregnancy, and that the mRNA levels of follistatin (Fst) (its ligand protein) are high in cycling rats and at the beginning of pregnancy and diminish thereafter, having the acini higher levels of expression. Our data show that activin betaB-subunit, follistatin and ActRIIA and IIB transcripts are expressed in rat mammary gland at appropriate times and locations during development, allowing an interplay that might regulate activin action on growth and differentiation of the gland.
Assuntos
Ativinas/fisiologia , Folistatina/biossíntese , Subunidades beta de Inibinas/fisiologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Receptores de Ativinas/biossíntese , Receptores de Ativinas/genética , Ativinas/genética , Ativinas/farmacologia , Animais , Caseínas/biossíntese , Caseínas/genética , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Folistatina/genética , Folistatina/farmacologia , Regulação da Expressão Gênica , Subunidades beta de Inibinas/biossíntese , Subunidades beta de Inibinas/genética , Subunidades beta de Inibinas/farmacologia , Inibinas/biossíntese , Inibinas/genética , Lactação/fisiologia , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/fisiologia , Gravidez , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta/farmacologiaRESUMO
A oxidação da lipoproteína da baixa densidade (LDL) é um evento crucial na aterogênese. Os antioxidantes, que protegem a LDL contra a oxidação, potencialmente, podem exercer efeitos benéficos quanto ao desenvolvimento e progressão da aterosclerose. O objetivo principal deste trabalho foi avaliar o efeito das isoflavonas da soja, que apresentam atividade antioxidante, sobre a formação da fração da LDL oxidada in vivo, denominada LDL- e anticorpos anti-LDL-, a colesterolemia e a formação de lesões ateroscleróticas, em coelhos com hipercolesterolemia induzida por uma dieta contendo caseína. Coelhos machos da raça Nova Zelândia (n=15) foram divididos randomicamente em três grupos, a saber: grupo X, que consumiu...
Assuntos
Animais , Coelhos , Caseínas/biossíntese , Caseínas/metabolismo , Glycine max/metabolismo , Hipercolesterolemia , Isoflavonas , Coleta de Amostras Sanguíneas , Cromatografia Líquida/métodos , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática , Imunoensaio , Lipoproteínas HDL/análise , Lipoproteínas LDL/análise , Lipoproteínas VLDL/análiseRESUMO
La diferenciación funcional del epitelio mamario requiere de hormonas específicas, factores de crecimiento y señales del medio ambiente celular. Estas últimas consisten en comunicación de sus células con la matriz extracelular e interacciones célula-célula, las cuales determinan un intrincado mecanismo que se traduce, finalmente, en eventos que conllevan a la secreción láctea. Células epiteliales mamarias de rata HC11, un subclon de la línea celular COMMA-1D de glándula mamaria en mitad de la preñez, fueron cultivadas en presencia de hormonas lactogénicas, insulina, dexametasona y prolactina y estimuladas con el factor de crecimiento epidérmico. En estas condiciones, las células se diferencian sintetizando ß caseína, demostrada mediante inmuno-blotting y filamentos intermediarios de citoqueratina, los cuales son evidenciados en micrografías electrónicas y con reacción inmuno-citoquímica con anticuerpos anticitoqueratina. Además, en estas células normales-proliferantes y diferenciales, fue realizado un estudio morfométrico, cuyos resultados correlacionan adecuadamente con los eventos morfológicos y bioquímicos observados en esta diferenciación del epitelio mamario de rata, en cultivo
Assuntos
Animais , Ratos , Mama/citologia , Diferenciação Celular , Análise de Variância , Western Blotting , Caseínas/biossíntese , Meios de Cultura , Epitélio/citologia , Microscopia EletrônicaRESUMO
HC11 is a normal mouse mammary epithelial cell line that requires certain growth factors, such as EGF or bFGF, to respond optimally to lactogenic hormones and produce the differentiation marker beta-casein. Growth in insulin (Ins) or PDGF does not produce cells competent to respond to lactogenic hormones. Here we show that competency for differentiation is due at least in part to the modulation of extracellular matrix components. In particular we have studied laminin and tenascin. EGF alters endogenous laminin assembly. In addition, promotion of competency can be partially mimicked by plating HC11 cells on the E8 laminin fragment, which is able to induce lactogenic responsiveness in cells grown in the absence of EGF or bFGF. The production and assembly of tenascin is also dependent upon the growth conditions of the HC11 cells. EGF- or bFGF-grown competent cells produce tenascin but do not assemble it at the extracellular matrix as efficiently as Ins- or PDGF-grown, non-competent cells. This alteration apparently leads to a change in the cellular microenvironment that supports beta-casein production. In addition, when competent cells are plated on dishes coated with tenascin, lactogenic hormone induction of beta-casein is inhibited. The data suggest that tenascin assembly and beta-casein production are opposing features of a coordinated differentiation program of HC11 cells.