RESUMO
The epidermis must be protected against excess apoptotic cell death in response to ultraviolet-B (UV-B) irradiation. p53 is known to be critical for this protection. Although the p53 family member DeltaNp51B/DeltaNp63alpha (an N terminal-deleted form of p51/p63) is abundantly expressed in keratinocytes, its contribution to UV-B-dependent apoptosis is largely unknown. We found that, after a transient increase, DeltaNp51B is downregulated in UV-B-irradiated keratinocytes undergoing apoptosis, whereas p53 is upregulated with delayed kinetics. Furthermore, the reduction of DeltaNp51B by small interfering RNAs augmented UV-B-dependent apoptosis in keratinocytes, indicating that DeltaNp51B blocks keratinocyte apoptosis. Although the exogenous expression of DeltaNp51B in keratinocytes did not further block the UV-B-dependent apoptosis, to our surprise the expression of TAp51B (an isoform with a full NH(2)-terminal transactivation domain that is structurally and functionally similar to p53) decreased apoptosis significantly. The blockade of keratinocyte apoptosis by the p51 was dependent on the phosphorylation of Akt, resulting in the activation of a survival pathway. Thus, in addition to its indispensable roles in epithelial development, p51 acts in adult cells to protect the epidermis against UV-B irradiation by preventing excess depletion of keratinocytes.
Assuntos
Epiderme/efeitos da radiação , Proteínas Nucleares/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tolerância a Radiação , Raios Ultravioleta , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/genética , Células Cultivadas , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Camundongos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , RNA Interferente Pequeno/farmacologiaRESUMO
p53 homologue, p51/p63, predominantly expressed in keratinocyte stem cells, is indispensable for the formation of epidermis. Notch1, another such gene indispensable for the process, induces growth arrest and differentiation in keratinocytes. We found that exogenous expression of DeltaNp51B (DeltaNp63alpha), one of the isoforms of p51 specifically expressed in basal keratinocytes, blocked Notch 1-dependent growth arrest and differentiation in mouse keratinocytes by inhibiting p21 expression and maintaining integrins expression. Furthermore, DeltaNp51B by itself was found to have ability to induce expression of integrin alpha6beta4, which promotes attachment of basal cells to basal membrane thereby keeping the cells in immature state. Therefore, we conclude that DeltaNp51B expression warrants integrin expression even under the influence of Notch1 and that DeltaNp51B is a long-sought factor required to maintain basal cell keratinocytes immaturity by inhibiting Notch1 activity. We will postulate a plausible model explaining the maintenance of the squamous epithelium architectures as well as offering mechanistic explanations for pathological features of skin diseases, including cancers, psoriasis along with physiological wound healings.
Assuntos
Genes p53 , Queratinócitos/fisiologia , Fosfoproteínas/genética , Receptor Notch1/metabolismo , Transativadores/genética , Animais , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Camundongos , Isoformas de Proteínas/fisiologia , Células-Tronco/fisiologia , TransfecçãoRESUMO
The suprachiasmatic nucleus of the anterior hypothalamus is the center of an internal biological clock in mammals. Glutamate is the neurotransmitter of retino-hypothalamic tract responsible for mediating the circadian actions of light in rodents. N-methyl-d-aspartate receptors, particularly NR2B subunit are reported to be principally involved in photic resetting of the biological clock in vivo and in slice culture. But, the precise cellular mechanisms of the resetting are not elucidated, because no adequate neuronal cell lines derived from the suprachiasmatic nucleus have been established. We established a neuronal cell line, N14.5, derived from the suprachiasmatic nucleus of a transgenic rat harboring the temperature-sensitive simian virus 40 large T-antigen gene. When the cells were cultured at 39 degrees C, the morphological features were turned fibroblastic into neuronal round cell body with neurite extensions. These cells showed immunoreactivities for neuronal markers (betaIII-tubulin, microtubule-associated protein 2 and TAU2) and as well as for vasoactive intestinal peptide which is expressed in the ventrolateral region of the suprachiasmatic nucleus. The cells expressed N-methyl-d-aspartate receptors, particularly NR1 and NR2B subunits as revealed by quantitative PCR. N-methyl-d-aspartate activated phosphorylation of p44/42 mitogen-activated protein kinase and increased expression level of Per1 and Per2 mRNA. These results suggest that the N14.5 is a novel neuronal cell line derived from the ventrolateral region of the suprachiasmatic nucleus, and that N-methyl-d-aspartate receptors expressed in the cells are a functional receptor. The N14.5 cells may be a useful tool to elucidate numerous chronobiological processes, especially resetting mechanism induced by an external light signal.
Assuntos
Relógios Biológicos/genética , Ritmo Circadiano/genética , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Núcleo Supraquiasmático/metabolismo , Animais , Animais Geneticamente Modificados , Antígenos Transformantes de Poliomavirus/genética , Relógios Biológicos/efeitos da radiação , Biomarcadores/metabolismo , Proteínas de Ciclo Celular , Diferenciação Celular/fisiologia , Linhagem Celular , Ritmo Circadiano/efeitos da radiação , Regulação da Expressão Gênica/fisiologia , Vetores Genéticos/genética , Luz , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/efeitos da radiação , Proteínas Nucleares/genética , Proteínas Circadianas Period , Estimulação Luminosa , Ratos , Núcleo Supraquiasmático/citologia , Núcleo Supraquiasmático/efeitos da radiação , Fatores de Transcrição/genética , Transfecção/métodos , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
The successive stages of development from oligodendrocyte progenitor to mature oligodendrocyte have been investigated in detail by using stage-specific antibodies. However, no cell lines are available that show stepwise differentiation from oligodendrocyte progenitors to mature oligodendrocytes. Here we show the establishment of an immortalized oligodendrocyte cell line, OLP6, from adult transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. The OLP6 cells had a fibroblastic morphology and continuously proliferated at 33 degrees C. They displayed growth arrest and multipolar morphology when they were cultured at 39 degrees C. They express the oligodendrocytic markers O4, 2'-3'-cyclic-nucleotide 3'-phosphodiesterase, galactocerebroside and second endothelial differentiation gene receptor-2 at 39 degrees C. The OLP6 cells underwent apoptosis upon serum withdrawal at 39 degrees C. Lysophosphatidic acid inhibited this apoptosis and promoted the expression of myelin basic protein. These results demonstrate that the activation of endothelial differentiation gene receptor-2 exerts anti-apoptosis and myelinogenesis effects on the OLP6 cells. Taken together, the OLP6 cells in the late oligodendrocyte progenitor stage can progress to the immature oligodendrocyte stage by shifting culture temperature. Furthermore, lysophosphatidic acid promoted the maturation of OLP6 cells in the immature oligodendrocyte stage. Such OLP6 cells should provide a potent model system for studying the precise mechanism involved in stepwise differentiation of oligodendrocytes.
Assuntos
Linhagem Celular Transformada , Senescência Celular , Oligodendroglia/citologia , Oligodendroglia/fisiologia , Células-Tronco/citologia , Animais , Animais Geneticamente Modificados , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Viral , Imuno-Histoquímica , Lisofosfolipídeos/farmacologia , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/fisiologia , Oligodendroglia/metabolismo , Ratos , Receptores de Ácidos Lisofosfatídicos/metabolismo , Vírus 40 dos Símios/fisiologia , TemperaturaRESUMO
Development of the musculoskeletal system requires coordinated formation of distinct types of tissues, including bone, cartilage, muscle, and tendon. Compared to muscle, cartilage, and bone, cellular and molecular bases of tendon development have not been well understood due to the lack of tendon cell lines. The purpose of this study was to establish and characterize tendon cell lines. Three clonal tendon cell lines (TT-E4, TT-G11, and TT-D6) were established using transgenic mice harboring a temperature-sensitive mutant of SV40 large T antigen. Proliferation of these cells was significantly enhanced by treatment with bFGF and TGF-beta but not BMP2. Tendon phenotype-related genes such as those encoding scleraxis, Six1, EphA4, COMP, and type I collagen were expressed in these tendon cell clones. In addition to tendon phenotype-related genes, expression of osteopontin and Cbfal was observed. These clonal cell lines formed hard fibrous connective tissue when implanted onto chorioallantoic membrane in ovo. Furthermore, these cells also formed tendon-like tissues when they were implanted into defects made in patella tendon in mice. As these tendon cell lines also produced fibrocartilaginous tissues in tendon defect implantation experiments, mesenchymal stem cell properties were examined. Interestingly, these cells expressed genes related to osteogenic, chondrogenic, and adipogenic lineages at low levels when examined by RT-PCR. TT-G11 and TT-E4 cells differentiated into either osteoblasts or adipocytes, respectively, when they were cultured in cognate differentiation medium. These observations indicated that the established tendon cell line possesses mesenchymal stem cell-like properties, suggesting the existence of mesenchymal stem cell in tendon tissue.
Assuntos
Linhagem Celular , Mesoderma/citologia , Células-Tronco Pluripotentes/citologia , Tendões/citologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Antígenos Transformantes de Poliomavirus/genética , Bovinos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Transplante de Células , Embrião de Galinha , Córion/crescimento & desenvolvimento , Córion/transplante , Células Clonais , Colágeno Tipo I/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Camundongos , Camundongos Transgênicos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteopontina , Sialoglicoproteínas/metabolismo , Temperatura , Tendões/embriologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
A Leydig cell line, TTE1, has been established from transgenic mice harboring a temperature-sensitive simian virus 40 (tsSV40) large T-antigen gene. The cells grew at a permissive temperature (33 degrees C), but growth was markedly prevented at a nonpermissive temperature (39 degrees C). T-antigen was expressed in the nuclei at 33 degrees C but disappeared at 39 degrees C, indicating that the cells show a temperature-sensitive growth phenotype reflected by the tsSV40 large T-antigen. TTE1 cells did not show any colony-forming activity in soft agar and form tumors in subcutaneous tissue in nude mice, indicating that the cells were not transformed. Alkaline phosphatase and 3beta-hydroxysteroid dehydrogenase (HSD) activities or expression of cytokeratin and vimentin were observed. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that TTE1 cells expressed mRNAs encoding 17beta-HSD types 1 and 3, and inhibin-alpha. The cells with unique characteristics, therefore, should serve useful model study the function of Leydig cell.
Assuntos
Antígenos Virais de Tumores/imunologia , Células Intersticiais do Testículo/citologia , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Masculino , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , TemperaturaRESUMO
To characterize acidic amino acid transport in type 2 astrocytes, we established conditionally immortalized rat astrocyte cell lines (TR-AST) from newly developed transgenic rats harboring temperature-sensitive SV40 large T-antigen gene. TR-AST exhibited positive immunostaining for anti-GFAP antibody and A2B5 antibody, characteristics associated with type 2 astrocytes, and expressed glutamine synthetase. Acidic amino acid transporters, GLT-1 and system xc-, which consists of xCT and 4F2hc, were expressed in all TR-ASTs by RT-PCR. On the other hand, GLAST expression was found in TR-AST3 and 5. The characteristics of [3H]L-glutamic acid (L-Glu) uptake by TR-AST5 include an Na+-dependent and Na+-independent manner, concentration-dependence, and inhibition by L-aspartic acid (L-Asp) and D-aspartic acid (D-Asp). The corresponding Michaelis-Menten constants for the Na+-dependent and Na+-independent process were 36.3 microM and 155 microM, respectively. [3H]L-Asp and [3H]D-Asp uptake by TR-AST5 had an Na+-dependent and Na+-independent manner. This study demonstrated that GLT-1, system xc-, and GLAST were expressed in TR-AST, which has the characteristics of type 2 astrocytes and is able to transport acidic amino acids.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Aminoácidos/metabolismo , Astrócitos/citologia , Astrócitos/metabolismo , Linhagem Celular Transformada , Animais , Animais Geneticamente Modificados , Antígenos Transformantes de Poliomavirus/genética , Transporte Biológico Ativo , Proteínas de Transporte/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Glutamato-Amônia Ligase/metabolismo , Imuno-Histoquímica , Cinética , Masculino , Ratos , Sódio/metabolismo , TemperaturaRESUMO
To understand the gene regulation involved in the development of single-positive (SP) thymocytes, we generated transgenic mice in which the AML1 transcription factor is overexpressed. In these mice the number of CD8 SP thymocytes was greatly increased, and this continued to be true even when MHC class I was absent. This promotion to the CD8 SP lineage was not, however, observed when both class I and class II were absent. Furthermore, even thymocytes carrying MHC class II-restricted TCR differentiated into the CD8 SP lineage when AML1 was overexpressed. The selected CD8 SP cells were, however, unable to mature, as judged by the expression level of heat-stable Ag. Thus, overexpression of AML1 is able to skew class II-restricted thymocytes into the CD8 SP lineage, but not to drive the maturation of resulting selected CD8 SP cells.
Assuntos
Antígenos CD8/análise , Linhagem da Célula , Proteínas de Ligação a DNA/fisiologia , Proteínas Proto-Oncogênicas , Linfócitos T/fisiologia , Fatores de Transcrição/fisiologia , Animais , Antígenos CD4/análise , Subunidade alfa 2 de Fator de Ligação ao Core , Antígenos de Histocompatibilidade Classe I/fisiologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Camundongos , Camundongos TransgênicosRESUMO
Progestins are known to suppress the growth of normal human endometrial glands and endometrial carcinomas possessing PRs. To elucidate the molecular mechanisms of progestin-induced growth inhibition, the expression and functional involvement of p27Kip1 (p27), a cyclin-dependent-kinase inhibitor, was investigated using cultured normal endometrial glandular cells and endometrial carcinoma cell lines (Ishikawa; PR-positive, KLE; PR-negative). Growth of the normal endometrial glandular cells and Ishikawa cells was suppressed by treatment with progesterone and medroxyprogesterone acetate, respectively, in association with an increase in p27 protein expression. Immunoprecipitation revealed that progestins accelerated the complex formation of p27 and cdk2 in both types of cells. However, treatment with progestins did not show any marked alterations in the mRNA expression of p27 in either normal glandular cells or Ishikawa cells. On the other hand, p27 protein degradation experiments indicated that treatment with progesterone and medroxyprogesterone acetate prolonged the degradation time of the normal endometrial glandular cells and Ishikawa cells, respectively. Forced expression of the p27 protein using a p27 expression plasmid reduced the growth activity of normal endometrial glandular cells. These findings suggest that p27 is functionally involved in progestin-induced growth suppression of normal and malignant endometrial epithelial cells and that up-regulation of the p27 protein by progestins possibly occurs via posttranslational mechanisms.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Neoplasias do Endométrio/fisiopatologia , Endométrio/fisiologia , Neoplasias Epiteliais e Glandulares/fisiopatologia , Proteínas Supressoras de Tumor , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27 , Neoplasias do Endométrio/patologia , Endométrio/citologia , Feminino , Humanos , Neoplasias Epiteliais e Glandulares/patologia , Progestinas/farmacologia , Progestinas/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
If all individual cell types of the body could be clonally isolated and stocked, similar to cDNA or genomic DNA libraries, they would be invaluable for studying the tissue and cellular functions. We developed a new method of establishing conditionally immortalized cell lines that retain differentiated cell functions similar to the original tissues, using temperature-sensitive (ts) simian virus 40 large tumor antigen gene transgenic animals. In this review the properties of such conditionally immortalized cell lines and their possible applications are discussed.
Assuntos
Linhagem Celular Transformada , Animais , Animais Geneticamente Modificados , Antígenos Transformantes de Poliomavirus/genética , Diferenciação Celular , Avaliação Pré-Clínica de Medicamentos , Biblioteca Gênica , Camundongos , Camundongos Mutantes , Modelos Biológicos , Ratos , Células-Tronco/fisiologiaRESUMO
After detachment from the stromal cells, hematopoietic stem cells are thought to differentiate to the cytokine-dependent stages where their growth and differentiation are promoted by these cytokines. To examine the stromal regulation of hematopoietic stem cells, we previously established a primitive hematopoietic stem-like cell line, THS119, whose growth was dependent on the bone marrow stromal cell line, TBR59, and from which IL-3- (THS119/IL-3) or IL-7- (THS119/IL-7) dependent cell lines were then generated. Using these cell lines, we examined the difference in signals mediated by the stromal cells and cytokines. The cytokine-dependent cell lines (THS119/IL-3 and THS119/IL-7) showed induction of STAT5 phosphorylation and target genes for STAT5 such as CIS, pim-1, p21 and bcl-xL upon addition of IL-3 or IL-7. IL-3 or IL-7 also induced STAT5 phosphorylation and STAT5 target genes of the stromal cell-dependent cell line, THS119, in the absence of stromal cells at levels similar to the cytokine-dependent cell lines. However, quite interestingly, TBR59 stromal cells could not induce STAT5 phosphorylation of THS119 cells, although they did induce STAT5 target genes in THS119 cells. In addition, the mRNAs for STAT5 target genes in THS119 cells on the stromal cells seemed to be more stable than those in the cytokine-dependent cell lines. Expression of the antiapoptotic genes bcl-2 and bcl-xL was higher in the stromal cell-dependent cell line than in the cytokine-dependent cell lines. These results suggested that stromal cells and cytokines may provide different signals for growth and differentiation of the hematopoietic cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interleucina-3/metabolismo , Interleucina-7/metabolismo , Proteínas do Leite , Transdução de Sinais , Células Estromais/metabolismo , Transativadores/metabolismo , Divisão Celular , Linhagem Celular , Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Interleucina-3/farmacologia , Interleucina-7/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fator de Transcrição STAT5 , Células Estromais/fisiologia , Proteína X Associada a bcl-2RESUMO
PURPOSE: To establish and characterize a choroid plexus epithelial cell line (TR-CSFB) from a new type of transgenic rat harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat). METHODS: Choroid plexus epithelial cells were isolated from the Tg rat and cultured on a collagen-coated dish at 37 degrees C during the first period of 3 days. Cells were subsequently cultured at 33 degrees C to activate large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells using a penicillin cup. RESULTS: Five immortalized cell lines of choroid plexus epithelial cells (TR-CSFB 1 approximately 5) were obtained from two Tg rats. These cell lines had a polygonal cell morphology, expressed the typical choroid plexus epithelial cell marker, transthyretin, and possessed Na+, K+-ATPase on their apical side. TR-CSFBs cells expressed a large T-antigen and grew well at 33 degrees C with a doubling-time of 35 approximately 40 hr. [3H]-L-Proline uptake by TR-CSFB cells took place in an Na+-dependent, ouabain-sensitive, energy-dependent, and concentration-dependent manner. It was also inhibited by alpha-methylaminoisobutylic acid, suggesting that system A for amino acids operates in TR-CSFB cells. When [3H]-L-proline uptake was measured using the Transwell device, the L-proline uptake rate following application to the apical side was five-fold greater than that following application to the basal side. In addition, both Na+-dependent and Na+-independent L-glutamic acid (L-Glu) uptake processes were present in TR-CSFB cells. CONCLUSIONS: Immortalized choroid plexus epithelial cell lines were successfully established from Tg rats and have the properties of choroid plexus epithelial cells, and amino acid transport activity was observed in vivo.
Assuntos
Aminoácidos/metabolismo , Barreira Hematoencefálica/fisiologia , Linhagem Celular Transformada/metabolismo , Plexo Corióideo/citologia , Plexo Corióideo/metabolismo , Células Epiteliais/metabolismo , Animais , Animais Geneticamente Modificados , Antígenos Transformantes de Poliomavirus/metabolismo , Transporte Biológico/fisiologia , Ácido Glutâmico/metabolismo , Masculino , Pré-Albumina/metabolismo , Prolina/metabolismo , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
PURPOSE: Postneonatal neovascularization is thought to result exclusively from the proliferation, migration, and remodeling of fully differentiated endothelial cells (ECs). Recently, it has been reported that bone marrow contains cells which can differentiate into ECs and contribute to neoangiogenesis in adult species. In this study, we tried to establish conditionally immortalized endothelial cell lines (TR-BME) derived from rat bone marrow. METHODS: Mononuclear cells were isolated and differentiated into ECs at 37 degrees C from the bone marrow of a transgenic rat harboring temperature-sensitive SV40 large T-antigen (ts T-Ag) gene. Then, the cells were transferred and incubated at 33 degrees C, a permissive temperature for ts T-Ag. Expression of vascular endothelial growth factor (VEGF) receptor (VEGFR)-1, 2, Tie-1, 2 and von Willebrand factor (VWF) were assayed by reverse transcriptase-mediated polymerase chain reaction (RT-PCR). RESULTS: We have established three cell lines incorporating 1,1'-dioctadecyl-3,3,3',3-tetramethylindo-carbocyanine perchlorate (DiI-Ac-LDL) with a spindle shape. One of these, clone 2, strongly expressed VEGFR-2, and weakly expressed VEGFR-1 and VWF. In contrast, clone 8 showed strong expression of Tie-1, 2, and VWF, and weak expression of VEGFR-1,2. All markers were expressed strongly in clone 3. CONCLUSIONS: These data confirm that the above three TR-BME cells are novel ECs derived from bone marrow progenitors.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Linhagem Celular/citologia , Linhagem Celular/metabolismo , Endotélio/citologia , Endotélio/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Animais , Animais Geneticamente Modificados , Antígenos Transformantes de Poliomavirus/metabolismo , Técnicas de Cultura de Células/métodos , Feminino , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Receptores Proteína Tirosina Quinases/metabolismo , Receptor de TIE-1 , Receptor TIE-2 , Receptores de Superfície Celular/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de TIE , Receptores de Fatores de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fator de von Willebrand/metabolismoRESUMO
Complete understanding of the physiology and pathology of the cartilage is essential to establish treatments for a variety of cartilage disorders and defects such as rheumatoid arthritis, congenital malformations, and tumors of cartilage. Although synthetic materials have been used in many cases, they possess inherent problems including wear of the materials and low mechanical strength. Autograft has been considered very effective to overcome these problems. However, the limitation of the transplant volume is a major problem in autograft to be overcome. The costal cartilage is the most serious candidate for donor site transplantation, since it is the largest permanent hyaline cartilage in the body. To investigate the possibility using the costal cartilage as a transplant source, we have established and characterized three mouse chondrocyte cell lines (MCC-2, MCC-5, and MCC-35) derived from the costal cartilage of 8-week-old male SV40 large T-antigen transgenic mice. At confluence, all the cell lines formed nodules that could be positively stained with alcian blue (pH 2.5). The size of nodules gradually increased during culturing time. After 2 and 6 weeks of culture, RT-PCR analysis demonstrated that all three cell lines expressed mRNA from the cartilage-specific genes for type II collagen, type XI collagen, aggrecan, and link protein. Furthermore, type X collagen expression was detected in MCC-5 and MCC-35 but not in MCC-2. Any phenotypic changes were not observed over 31 cell divisions. Immunocytochemistry showed further that MCC-2, MCC-5, and MCC-35 produced cartilage-specific proteins type II collagen and type XI collagen, while in addition MCC-5 and MCC-35 produced type X collagen. Treatment with 1alpha, 25-dihydroxyvitamin D(3) inhibited cell proliferation and differentiation of the three cell lines in a dose-dependent manner. These phenotypic characteristics have been found consistent with chondrocyte cell lines established from cartilage tissues other than costal cartilage. In conclusion, costal cartilage shows phenotypic similarities to other cartilages, i.e., articular cartilage and embryonic limbs, suggesting that costal cartilage may be very useful as the donor transplantation site for the treatment of cartilage disorders. Furthermore, the cell lines established in this study are also beneficial in basic research of cartilage physiology and pathology.
Assuntos
Cartilagem/citologia , Condrócitos/metabolismo , Matriz Extracelular/fisiologia , Expressão Gênica/genética , Fosfatase Alcalina/metabolismo , Animais , Antígenos Transformantes de Poliomavirus/genética , Biomarcadores , Calcitriol/farmacologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular/metabolismo , Condrócitos/citologia , Colágeno/biossíntese , Masculino , Camundongos , Camundongos TransgênicosRESUMO
We established two gingival epithelial cell lines (GE1 and GE6), originating from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene. GE1 and GE6 grew at a permissive temperature (33 degrees C) in a pavement arrangement and solely formed multilayers that exhibited morphological features similar to those of the stratified oral epithelium, with neither the use of stromal equivalents nor feeder layers. Both GE cells underwent apoptosis at a non-permissive temperature (39 degrees C). Characteristic keratin peptides, keratin 4 and 13, for mucosal epithelium were obviously expressed in the suprabasal cells, and keratohyalin granules and involucrin were present in the surface flat cells in the multilayered culture. Keratin 10 (one of the markers for higher keratinized gingival epithelium) was rarely found in some uppermost cells, and filaggrin (a component of keratohyalin granules) appeared sparsely in uppermost desquamating cells in the older cultures. These observations indicated that GE1 and GE6 cells exhibited the phenotype characterizing nonkeratinized sulcular epithelium, which possessed the potency undergoing keratinization in such highly stratified cultures as oral gingival epithelium. GE cells increased the expression levels of mRNA of interleukin-1beta and tumor necrosis factor alpha by the stimulation of lipopolysaccharide and extracellular substances of oral streptococci. The GE cell lines thus could serve as an excellent experimental system for further studies on the physiology of gingival epithelium and corresponding diseases, such as periodontal disease, epithelial hyperplasia, and gingival tumors.
Assuntos
Técnicas de Cultura de Células/métodos , Células Epiteliais/fisiologia , Gengiva/citologia , Animais , Antígenos Virais/genética , Apoptose , Proteínas de Bactérias/farmacologia , Linhagem Celular Transformada , Células Clonais , Citocinas/biossíntese , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Proteínas Filagrinas , Gengiva/metabolismo , Gengiva/fisiologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Proteínas de Filamentos Intermediários/biossíntese , Queratinas/biossíntese , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Precursores de Proteínas/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus 40 dos Símios/imunologiaRESUMO
Primordial germ cells (PGCs) in mice have been recognized histologically as alkaline phosphatase (AP) activity-positive cells at 7.2 days post coitum (dpc) in the extra-embryonic mesoderm. However, mechanisms regulating PGC formation are unknown, and an appropriate in vitro system to study the mechanisms has not been established. Therefore, we have developed a primary culture of explanted embryos at pre- and early-streak stages, and have studied roles of cell and/or tissue interactions in PGC formation. The emergence of PGCs from 5.5 dpc epiblasts was observed only when they were co-cultured with extra-embryonic ectoderm, which may induce the conditions required for PGC formation within epiblasts. From 6.0 dpc onwards, PGCs emerged from whole epiblasts as did a fragment of proximal epiblast that corresponds to the area containing presumptive PGC precursors without neighboring extra-embryonic ectoderm and visceral endoderm. Dissociated epiblasts at these stages, however, did not give rise to PGCs, indicating that interactions among a cluster of a specific number of proximal epiblast cells is needed for PGC differentiation. In contrast, we observed that dissociated epiblast cells from a 6.5-b (6.5+15-16 hours) to 6.75 dpc embryo that had undergone gastrulation gave rise to PGCs. Our results demonstrate that stage-dependent tissue and cell interactions play key roles in PGC determination.
Assuntos
Comunicação Celular , Diferenciação Celular , Células Germinativas/citologia , Animais , Biomarcadores , Contagem de Células , Linhagem da Célula , Técnicas de Cocultura , Técnicas de Cultura/métodos , Ectoderma/citologia , Desenvolvimento Embrionário e Fetal , Feminino , Fibronectinas/metabolismo , Gástrula/citologia , Idade Gestacional , Imuno-Histoquímica , Masculino , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Camundongos EndogâmicosRESUMO
The objective of this study was to establish and characterize a retinal capillary endothelial cell line (TR-iBRB) from a newly developed transgenic rat harboring the temperature-sensitive simian virus 40 (SV 40) large T-antigen gene (Tg rat). Retinal capillary endothelial cells were isolated from a Tg rat and cultured in collagen-coated dishes at 37 degrees C for a period of 48 hr. Cells were subsequently cultured at 33 degrees C to activate the large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells. Nine immortalized cell lines of retinal capillary endothelial cells (TR-iBRB1 approximately 9) were obtained from a Tg rat. These cell lines had a spindle-fiber shape morphology, expressed the typical endothelial marker, von Willebrand factor, and internalized acetylated-low density lipoprotein. Moreover, vascular endothelial growth factor (VEGF) receptor-2 was expressed in TR-iBRBs. TR-iBRBs expressed a large T-antigen and grew well at 33 degrees C with a doubling time of 19-21 hr. In contrast, cells did not grow at 37 and 39 degrees C due to the reduced expression of large T-antigen, supporting temperature-dependent cell growth. TR-iBRBs expressed GLUT1 and exhibited 3- O -methyl- D -glucose (3-OMG) uptake activity. This 3-OMG uptake was saturable with a Michaelis-Menten constant of 5.56 +/- 0.51 m M and a maximum uptake rate of 45.3 +/- 2.6 nmol min(-1) mg protein(-1). P-Glycoprotein, with a molecular weight of approximately 180 KDa, was expressed in TR-iBRBs. In addition, mdr 1a, mdr 1b and mdr 2 were detected in TR-iBRB2 using RT-PCR. In conclusion, conditionally immortalized retinal capillary endothelial cell lines were established from a transgenic rat harboring the temperature-sensitive SV 40 large T-antigen gene and these lines were shown to exhibit the properties of retinal capillary endothelial cells.
Assuntos
Barreira Hematorretiniana , Endotélio Vascular/patologia , Células Tumorais Cultivadas , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Animais , Animais Geneticamente Modificados , Antígenos Virais de Tumores/análise , Antígenos Virais de Tumores/genética , Western Blotting , Capilares , Divisão Celular , Separação Celular , Genes MDR , Transportador de Glucose Tipo 1 , Temperatura Alta , Modelos Animais , Proteínas de Transporte de Monossacarídeos/análise , Ratos , Receptores Proteína Tirosina Quinases/análise , Receptores de Fatores de Crescimento/análise , Receptores de Fatores de Crescimento do Endotélio Vascular , Vasos Retinianos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The growth, differentiation, and death/survival of spermatogonia are precisely regulated for the proper production of spermatozoa. We have previously shown that Bcl-2 ectopically expressed in spermatogonia caused the inhibition of normal spermatogonial apoptosis and the subsequent failure of differentiation in transgenic mice. In addition, the growth of spermatogonial stem cells seemed to be temporally arrested in the transgenic mice. In the present study, we attempted to examine whether the abnormality of spermatogonia described above was caused by Bcl-2 misexpression in the spermatogonia or by an abnormal spermatogenic environment of the transgenic mice. We transplanted testicular cells of transgenic mice to seminiferous tubules of W/Wv mice in which transplanted normal testicular cells can undergo spermatogenesis. We found that the transplanted spermatogonia of the transgenic mice reproduced a series of abnormal changes including temporal growth arrest of spermatogonial stem cells and abnormal accumulation of spermatogonia in tubules, which were also observed in the testes of the transgenic mice. The results indicated that Bcl-2 inhibited apoptosis of spermatogonia and growth of spermatogonial stem cells in a cell-intrinsic manner. We also cultured testicular cells of transgenic mice and found that the spermatogonia of the transgenic mice were better able to survive than were those of wild-type mice but that their differentiation was not affected. The result suggested that failure of differentiation of the accumulated spermatogonia in the transgenic testes is not due to the abnormality of the bcl-2 misexpressing spermatogonia, but may be caused by extrinsic problems including improper interaction of spermatogonia with supporting cells.
Assuntos
Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Espermatogênese/fisiologia , Espermatogônias/citologia , Trometamina/análogos & derivados , Animais , Diferenciação Celular , Sobrevivência Celular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Espermatozoides/citologia , Células-Tronco/citologia , Testículo/citologia , Fatores de TempoRESUMO
Bone marrow stromal cell lines (TBR cell lines) established from temperature-sensitive Simian Virus 40 T-antigen gene transgenic mice exhibited myogenic, osteogenic, and adipogenic differentiation. The effect of oncostatin M (OSM) on such mesenchymal cell differentiation of marrow stromal cell lines was examined. One of those stromal cell lines, TBRB, differentiated into skeletal muscle, and its differentiation was stimulated by OSM, whereas differentiation of TBR10-1 into smooth muscle was inhibited by OSM. TBR31-2 is a bipotent progenitor for adipocytes and osteoblasts, and OSM stimulated osteogenic differentiation while inhibiting adipogenic differentiation. On the other hand, TBR cell lines exhibited various potentials for supporting hematopoiesis in culture. When hematopoietic progenitor cells were cocultured with OSM-stimulated stromal cell lines, TBR10-1 and TBR31-2 exhibited enhanced hematopoietic supportive activity. As responsible molecules for stromal cell dependent hematopoiesis, expression of stem cell factor (SCF) (a ligand of c-Kit), vascular cell adhesion molecule (VCAM-1) (a ligand of VLA-4), and secretion of interleukin (IL)-6 were increased by OSM. OSM affected mesenchymal cell differentiation and promoted the hematopoietic supportive activity of marrow stromal cell lines. As OSM production is induced by cytokines from hematopoietic cells, OSM may be a key factor in mutual regulation between hematopoietic cells and stromal cells in the bone marrow. OSM may play a role as a regulator in maintaining the hematopoietic microenvironment in marrow by coordinating mesenchymal differentiation.