RESUMO
The understanding of the mechanisms that regulate gene expression to establish differentiation programs and determine cell lineages, is one of the major challenges in Developmental Biology. Besides the participation of tissue-specific transcription factors and epigenetic processes, the role of general transcription factors has been ignored. Only in recent years, there have been scarce studies that address this issue. Here, we review the studies on the biological activity of some TATA-box binding protein (TBP)-associated factors (TAFs) during the proliferation of stem/progenitor cells and their involvement in cell differentiation. Particularly, the accumulated evidence suggests that TAF4, TAF4b, TAF7L, TAF8, TAF9, and TAF10, among others, participate in nervous system development, adipogenesis, myogenesis, and epidermal differentiation; while TAF1, TAF7, TAF15 may be involved in the regulation of stem cell proliferative abilities and cell cycle progression. On the other hand, evidence suggests that TBP variants such as TBPL1 and TBPL2 might be regulating some developmental processes such as germ cell maturation and differentiation, myogenesis, or ventral specification during development. Our analysis shows that it is necessary to study in greater depth the biological function of these factors and its participation in the assembly of specific transcription complexes that contribute to the differential gene expression that gives rise to the great diversity of cell types existing in an organism. The understanding of TAFs' regulation might lead to the development of new therapies for patients which suffer from mutations, alterations, and dysregulation of these essential elements of the transcriptional machinery.
Assuntos
Proteína de Ligação a TATA-Box , Humanos , Diferenciação Celular/genética , Mutação , Proteínas Nucleares/genética , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/química , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Proteína de Ligação a TATA-Box/genética , AnimaisRESUMO
Acanthamoeba spp. are free-living amoebae with a worldwide distribution. These amoebae can cause granulomatous amoebic encephalitis and amoebic keratitis in humans. Proteases are considered virulence factors in pathogenic Acanthamoeba. The objective of this study was to evaluate the behavior of Acanthamoeba mauritaniensis, a nonpathogenic amoeba. We analyzed the cytopathic effect of A. mauritaniensis on RCE1(5â¯T5) and MDCK cells and compared it to that of Acanthamoeba castellanii. A partial biochemical characterization of proteases was performed in total crude extracts (TCE) and conditioned medium (CM). Finally, we evaluated the effect of proteases on tight junction (TJ) proteins and the transepithelial electrical resistance of MDCK cells. The results showed that this amoeba can induce substantial damage to RCE1(5T5) and MDCK cells. Moreover, the zymograms and Azocoll assays of amoebic TCE and CM revealed different protease activities, with serine proteases being the most active. Furthermore, A. mauritaniensis induced the alteration and degradation of MDCK cell TJ proteins with serine proteases. After genotyping this amoeba, we determined that it is an isolate of Acanthamoeba genotype T4D. From these data, we suggest that A. mauritaniensis genotype T4D behaves similarly to the A. castellanii strain.
Assuntos
Acanthamoeba/genética , Acanthamoeba/patogenicidade , Genótipo , Acanthamoeba/enzimologia , Animais , Cães , Células Epiteliais/parasitologia , Células Epiteliais/patologia , Células Madin Darby de Rim Canino , Serina Proteases/metabolismo , Proteínas de Junções Íntimas/metabolismoRESUMO
Vimentin (Vim), a cytoskeletal intermediate filament, is part of a naturally occurring reversible program, the Epithelial-Mesenchymal Transition (EMT), which converts epithelial cells into mesenchymal-like derivatives. Based on previous results showing that epithelial cells co-express Vim and keratin (Krt) as part of a cytoskeletal network which confers them a highly motile phenotype, we explored the role of Vim in rabbit corneal epithelial cells or RCE1(5T5) cells, an established model of corneal epithelial differentiation. Vim and keratin filaments were co-expressed in cells localized at the proliferative/migratory rim of the growing colonies, but not in basal cells from the center of the colonies nor at suprabasal cell layers. Flow cytometry and qPCR demonstrated that there was a decrease in Krt+ /Vim+ cell number and ΔNp63α expression when cells reached confluence and formed a 4-5 layered epithelium, while there was a concomitant increase of both Pax-6 expression and Krt+ /Vim- cells. Inhibition of cell proliferation with mitomycin C did not modify cell motility nor the expression of Vim. We studied the distribution and expression of α6 integrin, a protein also involved in cell migration. The results demonstrated that α6 integrin had a distribution which was, in part, co-linear with Vim at the proliferative/migratory rim of cell colonies, suggesting an indirect interaction between these proteins. Immunoprecipitation and immunostaining assays indicated that plectin might be mediating such interaction. These data suggest that Vim expression in corneal epithelium is found in a cell population composed of highly motile cells with a Vim+ /Krt+ /ΔNp63α+ /Pax-6low /α6 integrin+ phenotype. J. Cell. Physiol. 232: 818-830, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Diferenciação Celular , Movimento Celular , Células Epiteliais/citologia , Epitélio Corneano/citologia , Vimentina/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Integrina alfa6/metabolismo , Queratinas/metabolismo , Mitomicina/farmacologia , Plectina/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Coelhos , Proteínas Supressoras de Tumor/metabolismoRESUMO
Epithelial migration plays a central role in development, wound repair and tumor metastasis, but the role of intermediate filament in this important event is unknown. We showed recently that vimentin coexists in the same cell with keratin-KRT14 at the leading edge of the migrating epidermal cells, and knockdown of vimentin impaired colony growth. Here we demonstrate that vimentin co-localizes and co-immunoprecipitates with keratin-KRT14, and mutations in the -YRKLLEGEE- sequence of vimentin significantly reduced migration of the keratinocytes. Our data demonstrates that keratinocyte migration requires the interaction between vimentin and keratins at the -YRKLLEGEE- sequence at the helical 2B domain of vimentin. These findings have broad implications for understanding the roles of vimentin intermediate filaments in normal and neoplastic epithelial cells.
Assuntos
Células Epiteliais/citologia , Proteínas de Filamentos Intermediários/metabolismo , Queratina-14/metabolismo , Vimentina/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Movimento Celular , Células Cultivadas , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Inativação Gênica , Humanos , Proteínas de Filamentos Intermediários/química , Queratina-14/química , Queratina-14/imunologia , Queratinócitos/metabolismo , Ligação Proteica , Vimentina/química , Vimentina/genéticaRESUMO
Lipid droplets are dynamic organelles that store triglycerides and participate in their mobilization in adipose cells. These organelles require the reorganization of some structural components, the cytoskeleton, and the activation of lipogenic enzymes. Using confocal microscopy, we analyzed the participation of cytoskeletal components and two lipogenic enzymes, fatty acid synthase and glycerophosphate dehydrogenase, during lipid droplet biogenesis in differentiating 3T3-F442A cells into adipocytes. We show that subcortical actin microfilaments are extended at the basal side of the cells in parallel arrangement to the culture dish substrate, and that the microtubule network traverses the cytoplasm as a scaffold that supports the round shape of the mature adipocyte. By immunoprecipitation, we show that vimentin and perilipin1a associate during the early stages of the differentiation process for lipid droplet formation. We also report that the antibody against perilipin1 detected a band that might correspond to a modified form of the molecule. Finally, the cytosolic distribution and punctate organization of lipogenic enzymes and their co-localization in the proximity of lipid droplets suggest the existence of dynamic protein complexes involved in synthesis and storage of triglycerides. J. Cell. Biochem. 117: 2315-2326, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Citoplasma/metabolismo , Ácido Graxo Sintases/metabolismo , Glicerol-3-Fosfato Desidrogenase (NAD+)/metabolismo , Gotículas Lipídicas/fisiologia , Actinas/metabolismo , Adipócitos/citologia , Western Blotting , Células Cultivadas , Citoesqueleto/metabolismo , Ácido Graxo Sintases/genética , Imunofluorescência , Glicerol-3-Fosfato Desidrogenase (NAD+)/genética , Humanos , Lipogênese/fisiologia , Perilipina-1/genética , Perilipina-1/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tubulina (Proteína)/metabolismo , Vimentina/genética , Vimentina/metabolismoRESUMO
BACKGROUND: The bone nonunion is an important complication of bone fracture repair. The existing models developed on small animal species prevent using osteosynthesis materials designed to be implanted in human bones. The goal of this study was to develop a nonunion process in a noncritical segmental tibial defect in sheep, a species analogous in size to humans. MATERIALS AND METHODS: The animals were divided into two groups of four animals each. In Group 1 (experimental), the defect was created by surgically stripping the periosteum from the edges of a distal tibial osteotomy, keeping the edges 5 mm apart, and placing an incomplete O-shaped silicone ring in the gap. Group 2 (control) was intervened with a simple fracture at the distal end of the tibia. In both groups an interlocking nail was used as a fixation system. Over 8 wk after surgery, radiographs and histologic and histomorphometric analyses were performed. RESULTS: The control group showed a typical bone repair process. In contrast, the experimental group showed a fracture line with rounded edges and a scarce callus formation. The bone callus showed reduced amount of bone formation and large content of fibrous tissue (P=0.001). CONCLUSIONS: These results indicate that our model developed an atrophic nonunion in sheep, a species having multiple similarities to humans, such as weight, size, bone structure, and bone remodeling process.
Assuntos
Pinos Ortopédicos , Fixação Interna de Fraturas/instrumentação , Fixação Interna de Fraturas/métodos , Fraturas Mal-Unidas/cirurgia , Modelos Animais , Fraturas da Tíbia/cirurgia , Animais , Consolidação da Fratura , Fraturas Mal-Unidas/diagnóstico por imagem , Masculino , Osteogênese , Osteotomia , Periósteo/cirurgia , Radiografia , Ovinos , Tíbia/cirurgia , Fraturas da Tíbia/diagnóstico por imagemRESUMO
In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.
Assuntos
Decorina/genética , Regulação da Expressão Gênica , Queratinócitos/metabolismo , Células Cultivadas , Decorina/metabolismo , Epiderme/metabolismo , Epiderme/patologia , Humanos , Queratinócitos/patologia , Psoríase/metabolismo , Psoríase/patologiaRESUMO
To determine whether asymmetrical cell division takes place during growth and differentiation of corneal epithelial cells, we analyzed the expression of some proteins required for the correct execution of the asymmetric division in cultured RCE1-(5T5) cells, which mimic the differentiation of corneal epithelial cells. RT-PCR and immunostaining showed that Par-3, LGN (GPSM2), NuMA, and the mammalian homolog of inscuteable (Insc) are expressed by the cultured cells. Semi-quantitative RT-PCR demonstrated that Insc mRNA levels were stable throughout the experiment. Conversely, LGN and NuMA mRNAs increased slightly and steadily in proliferative cells, reaching a peak of about 20% above basal levels when cells were confluent. At later times, LGN and NuMA mRNAs decreased to become barely detectable when cells organized into a four-layered epithelium and expressed terminal phenotype as indicated by the highest expression of LDH-H mRNA. Cultivation under low Ca2+ conditions (0.09 mM) reduced about 50% Insc mRNA expression both in proliferating and confluent cultures, but did not affect the levels of LGN and NuMA mRNAs. Hence, asymmetric cell division seems to take place with a lower frequency in cells grown with low Ca2+ concentrations, in spite of the absence of stratification. Immunostaining experiments raise the possibility of an interaction between k3/K12 keratin cytoskeleton and Par-3. The results show for the first time the coordination between the expression of corneal epithelial cell differentiation and the expression of cell polarity machinery. They also suggest that asymmetric division does not depend on stratification; instead, it seems to be part of the differentiation program.
Assuntos
Diferenciação Celular , Divisão Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio Corneano/citologia , Células 3T3 , Animais , Cadaverina/farmacologia , Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Pax-6 is a regulatory gene with a major role during visual system development, but its association with corneal epithelial differentiation is not clearly established. Using the RCE1-(5T5) cell line, which mimics corneal epithelial differentiation, we analyzed Pax-6 biological role. Immunostaining of proliferating colonies and confluent sheets showed that Pax-6-positive cells were also K3 keratin-positive, suggesting that Pax-6 is expressed in differentiating cells. Pax-6 mRNA was barely expressed in early cell cultures; but after confluence, its levels raised up to fivefold as demonstrated by Northern blot and RT-qPCR. The raise in Pax-6 expression preceded for 9 h the increase in LDH-H and LDH-M mRNAs, previously shown as early markers of corneal epithelial cell differentiation. The full-length mRNAs encoding for the two major Pax-6 isoforms were found at very low levels in proliferating cells, and abundantly expressed in the confluent stratified epithelia; Pax-6 mRNA was 2- to 2.5-fold more abundant than Pax-6(5a) mRNA. The ectopic expression of Pax-6 or Pax-6(5a) decreased proliferative ability leading to the formation of abortive, non-proliferative colonies. In contrast, culture conditions that delay or block corneal epithelial cell differentiation reduced or inhibited the expression of Pax-6. Collectively, results show that Pax-6 is the earlier differentiation marker expressed by corneal epithelial cells, and open the possibility for a major role of Pax-6 as the main driver of the differentiation of corneal epithelial cells.
Assuntos
Diferenciação Celular/fisiologia , Córnea , Células Epiteliais/fisiologia , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Animais , Biomarcadores/metabolismo , Cadaverina/metabolismo , Linhagem Celular , Córnea/citologia , Córnea/fisiologia , Células Epiteliais/citologia , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Queratina-3/genética , Queratina-3/metabolismo , Lactato Desidrogenases/genética , Lactato Desidrogenases/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fenótipo , Coelhos , Proteínas Repressoras/genéticaRESUMO
Pre-adipose 3T3-F442A cells exposed to fetal bovine serum or human growth hormone (adipogenic medium) become irreversibly committed to differentiation into adipocytes within 24-36 h. We show now that the action of the serine-threonine kinase inhibitor staurosporine is much more rapid since its addition in non-adipogenic medium resulted in commitment to adipocyte differentiation within 4-6 h. During this period, glycogen synthase kinase 3beta was activated. Commitment depended on an increase in the intracellular calcium concentration that was modulated in part by a T-type calcium channel since mibefradil, amiloride, and NiCl(2), which are selective blockers of the T-type channels, partially inhibited adipose differentiation. Studies of the inhibitory action of retinoic acid showed that a period of time after exposure to St was required in order to stabilize the commitment to adipose differentiation. It was concluded that the commitment of the cells consists of two stages. Commitment is promoted during the first one, and during the second there is a stabilization which still can be destabilized by the addition of retinoic acid or other drugs. The commitment becomes stable after 40 h of staurosporine treatment, and can no longer be prevented by retinoic acid. The identification of these two stages of commitment makes it possible to analyze in further detail early molecular events of the process and the nature of any other participating genes.
Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Cálcio/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Estaurosporina/farmacologia , Adipócitos/efeitos dos fármacos , Animais , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta , Cloreto de Lítio/farmacologia , Camundongos , Fatores de Tempo , Tretinoína/farmacologiaRESUMO
Fibromodulin is a small leucine-rich proteoglycan that has a central role in the maintenance of collagen fibrils structure, and in regulation of TGF-beta biological activity. Although, it is mainly found in cartilage and tendon, little is known regarding the expression of the fibromodulin gene in other cell types. By RT-PCR, real time PCR and immunohistochemistry, we describe the expression of the fibromodulin gene and the presence of the protein in human epidermal keratinocytes (HEK), both in culture and in normal human epidermis. Our results show, for the first time, that fibromodulin gene is constantly expressed in HEK during culture time. Immunostaining showed that fibromodulin is located intracytoplasmically in basal and stratified keratinocytes of the growing colonies, confluent cultures, and epidermis in vivo. The expression and intracellular localization of fibromodulin in HEK is a new finding and opens new possible biological roles for the SLRP family.
Assuntos
Epiderme/metabolismo , Proteínas da Matriz Extracelular/genética , Expressão Gênica , Proteínas/genética , Proteoglicanas/genética , Células Cultivadas , Citoplasma/química , Citoplasma/metabolismo , Células Epidérmicas , Epiderme/química , Proteínas da Matriz Extracelular/análise , Proteínas da Matriz Extracelular/metabolismo , Fibromodulina , Humanos , Imuno-Histoquímica , Queratinócitos/química , Queratinócitos/metabolismo , Proteínas de Repetições Ricas em Leucina , Proteínas/análise , Proteínas/metabolismo , Proteoglicanas/análise , Proteoglicanas/metabolismoRESUMO
The immunogenicity of allogeneic cultured human epidermal keratinocytes (cHEKs) has been studied in several models with contradictory results. We studied human T-cell activation in an in vitro assay by incubating, for 4 and 24 hr, cHEK confluent sheets with human peripheral blood mononuclear cells (PBMC); parallel HEK cultures were incubated with interferon (IFN)-gamma to induce the expression of major histocompatibility complex (MHC) molecules before their interaction with PBMC. T-cell activation was evaluated by flow cytometry. T cells neither expressed the early and late activation markers CD69 and CD25, respectively, nor proliferated after incubation with the epidermal sheets, despite the IFN-gamma-induced expression of MHC and adhesion molecules in cHEKs. Interleukin (IL)-10 was detected in the medium from the co-cultured PBMC and HEK sheets, but not from HEK alone. The results suggest that HEKs are unable to stimulate T lymphocytes through secretion of cytokines that might contribute to the immunosuppressive effect in this in vitro model.
Assuntos
Epiderme/imunologia , Interleucina-10/imunologia , Queratinócitos/imunologia , Linfócitos T/imunologia , Comunicação Celular/imunologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Tolerância Imunológica/imunologia , Recém-Nascido , Ativação Linfocitária/imunologia , MasculinoRESUMO
Understanding of visual system function and the development of new therapies for corneal diseases and damages depend upon comprehension of the biological roles of the tissue. The in vitro cultivation of corneal epithelial cells and cell lines derived from them has become a powerful tool to analyze and understand such issues. Currently, researchers have developed well-defined and precisely described culture protocols and a collection of corneal epithelial cell lines. These cell lines have been obtained through different experimental approaches: (1) the ectopic expression of oncogenes, (2) the inactivation of p16 and p53 pathways and hTERT expression, and (3) the spontaneous establishment after serial cultivation of cells. The advantages or disadvantages for these approaches are discussed. In conclusion, the availability of several culture protocols and immortalized cell lines that express corneal epithelial phenotype will be useful for investigating issues such as gene regulation and tissue development, or for validating alternative methods in toxicology.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Epitélio Corneano/citologia , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Epitélio Corneano/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Oncogenes/fisiologiaRESUMO
We have studied the effects of interleukin-6 (IL-6) on human epidermal keratinocytes by using serum-free culture conditions that allow the serial transfer, differentiation, and formation of well-organized multilayered epithelia. IL-6 at 2.5 ng/ml or higher concentrations promoted keratinocyte proliferation, with an ED(50) of about 15 ng/ml and a maximum effect at 50 ng/ml. IL-6 was 10-fold less potent than epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) and supported keratinocyte growth for up to eight cumulative cell generations. IL-6-treated keratinocytes formed highly stratified colonies with a narrower proliferative/migratory rim than those keratinocytes stimulated with EGF or TGF-alpha; confluent epithelial sheets treated with IL-6 also underwent an increase in the number of cell layers. We also examined the effect of IL-6 on the keratin cytoskeleton. Immunostaining with anti-K16 monoclonal antibodies showed that the keratin network was aggregated and reorganized around cell nucleus and that this was not attributable to changes in keratin levels. This is the first report concerning the induction of the reorganization of keratin intermediate filaments by IL-6 in human epidermal keratinocytes.
Assuntos
Divisão Celular/efeitos dos fármacos , Interleucina-6/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/fisiologia , Queratinas/metabolismo , Anticorpos Monoclonais/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Meios de Cultura Livres de Soro , Relação Dose-Resposta a Droga , Células Epidérmicas , Fator de Crescimento Epidérmico/farmacologia , Humanos , Imuno-Histoquímica , Queratinócitos/ultraestrutura , Queratinas/ultraestrutura , Fator de Crescimento Transformador alfa/farmacologiaRESUMO
We describe a fast, sensitive, specific, and simple in vitro assay for GH biological activity, based on the differentiation of 3T3-F442A cells into adipocytes. The 3T3-F442A cells were directly plated at 1.5 x 10(4)cells/cm(2) in medium with or without various concentrations of human growth hormone (hGH). After 7 days, cells were lysed with buffer containing 0.5 % (v/v) Triton X-100, and adipose conversion was quantitated by the activity of the adipogenic enzyme glycerophosphate dehydrogenase. The assay is highly sensitive and specific for GH from different species. These culture conditions have shortened the time for the cells to undergo adipose differentiation, and they might also be useful to design and test drugs or agents that modify adipocyte differentiation or lipid metabolism, or for evaluation of cytotoxic and pharmacologic effects of drugs and other compounds.
Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/enzimologia , Técnicas Biossensoriais , Diferenciação Celular/efeitos dos fármacos , Glicerolfosfato Desidrogenase/metabolismo , Hormônio do Crescimento/análise , Hormônio do Crescimento/farmacologia , Células 3T3 , Adipócitos/citologia , Adipócitos/fisiologia , Animais , Relação Dose-Resposta a Droga , Ativação Enzimática , Glicerolfosfato Desidrogenase/análise , Camundongos , Reprodução , Sensibilidade e EspecificidadeRESUMO
We studied the commitment of 3T3-F442A cells during stimulation with adipogenic serum or growth hormone. Confluent 3T3-F442A preadipocytes were incubated with adipogenic medium for increasing times; the number of adipose clusters, GPDH activity, and lipid accumulation were evaluated. Results show that cell commitment took place during the first 24-36 h after stimulation under adipogenic conditions. Then, cultures underwent a 2-fold increase in total cell number through selective multiplication of committed cells, followed by a dramatic decrease in colony-forming ability and 300- to 1000-fold raise in GPDH activity. Cell commitment was not modulated by insulin, but this hormone stimulated clonal expansion of committed cells and lipogenesis. Commitment was inhibited by TNF-alpha at concentrations as low as 5 ng/ml, and by retinoic acid. The results show that TNF-alpha inhibits adipose conversion at two different levels: at concentrations as low as 5 ng/ml, it blocks commitment, and at concentrations of 100 ng/ml or higher the cytokine seems to block mitotic expansion and other steps of differentiation after cell commitment. The identification of a specific time for cell commitment would allow the study of the early genes that might regulate cell reprogramming into adipocytes.
Assuntos
Células 3T3/metabolismo , Adipócitos/metabolismo , Diferenciação Celular/fisiologia , Tempo de Reação/fisiologia , Células-Tronco/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Células 3T3/citologia , Células 3T3/efeitos dos fármacos , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/fisiologia , Meios de Cultivo Condicionados/farmacologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glicerol-3-Fosfato Desidrogenase (NAD+) , Glicerolfosfato Desidrogenase/metabolismo , Insulina/metabolismo , Insulina/farmacologia , Lipídeos/biossíntese , Camundongos , Tempo de Reação/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Tretinoína/metabolismo , Tretinoína/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
PURPOSE: To determine whether frozen cultured sheets of human allogeneic epidermal keratinocytes (CEAK) improved wound repair after experimental corneal ablation by photorefractive keratectomy (PRK). SETTING: Hospital "Luis Sanchez Bulnes" de la Asociación para Evitar la Ceguera en Mexico, I.A.P, and Department of Cell Biology, CINVESTAV-IPN, Mexico City, Mexico. METHODS: Transepithelial PRK was performed in the right eye of male albino rabbits to obtain a 112 microm deep and 6.0 mm diameter ablation zone. In 17 eyes, the ablations were covered with frozen CEAK; in 11 eyes, the ablations were covered with a disposable contact lens without the cultured sheets; and in the control group (13 eyes), the ablations were not covered. Subepithelial fibrosis and reepithelialization of the ablated zone were evaluated in serial paraffin-embedded tissue sections from all wounds. RESULTS: Treatment with CEAK reduced fibroblast proliferation and the inflammatory response beneath the ablated zone and produced better organization of the newly formed epithelium by eliminating significant hyperplasia or discontinuities in the periodic acid Shiff-stained basement membrane. It also led to accelerated reepithelialization. CONCLUSIONS: The use of frozen CEAK as a biologically active wound dressing improved tissue repair at 1 month in corneas ablated by transepithelial PRK in the male albino rabbit model. Treatment with CEAK could improve the outcome of PRK in humans.
Assuntos
Bandagens , Córnea/fisiopatologia , Epitélio Corneano/fisiopatologia , Congelamento , Queratinócitos/fisiologia , Ceratectomia Fotorrefrativa , Cicatrização/fisiologia , Animais , Células Cultivadas , Córnea/patologia , Células Epidérmicas , Epitélio Corneano/patologia , Humanos , Lasers de Excimer , Masculino , Período Pós-Operatório , CoelhosRESUMO
Lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) activities were studied during corneal epithelial growth and differentiation in cell culture. LDH and G-6-PDH activities increased up to 60 and 150-fold, respectively, when corneal epithelial cells constituted a differentiated four to five layered epithelium; these increases showed a similar time-course to the expression of K3 keratin. Immunostaining experiments showed that in growing colonies, LDH staining is stronger in those cells that are K3 positive; in contrast, in confluent four to five layered epithelia LDH and K3 were located in all cell layers, similar to the pattern found in frozen sections from rabbit central cornea. During growth and differentiation, the LDH isoenzyme set from corneal epithelial cells did not change; and it was different from those observed in cultured conjunctival, esophageal and epidermal cells. The augment in LDH activity was due to a 25-fold increase in the LDH-H mRNA and a 12-fold augment in LDH-M mRNA. A computer-assisted search led to identify AP2 and Sp1 binding sites in the LDH and G-6-PDH promoters, suggesting that their expression might share common regulatory mechanisms with the regulation of the differentiation-linked keratins. It is proposed that LDH may be an early marker of corneal epithelial differentiation, and its isozyme pattern could be distinctive from other epithelial cell lineages.
Assuntos
Células Epiteliais/enzimologia , Epitélio Corneano/enzimologia , L-Lactato Desidrogenase/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Células Epiteliais/ultraestrutura , Epitélio Corneano/ultraestrutura , Imunofluorescência , Expressão Gênica , Glucosefosfato Desidrogenase/metabolismo , Isoenzimas/metabolismo , Queratinas/metabolismo , L-Lactato Desidrogenase/genética , Masculino , RNA Mensageiro/genética , CoelhosRESUMO
In a murine model of full-thickness wounds, healing is stimulated by the application of human frozen cultured epidermal sheets. With immunofluorescence techniques, we studied, during this process, the spatial and temporal pattern of expression of: transforming growth factor-alpha (TGF-alpha); transforming growth factor-beta (TGF-beta) isoforms 1, 2, and 3; platelet-derived growth factor (PDGF); and the extracellular matrix proteins fibronectin, collagen IV, and tenascin. The growth factors, with the exception of PDGF, were found to be located in the frozen cultured sheet of keratinocytes before and after its application to the wound, whereas collagen IV and tenascin were deposited in the connective tissue under the frozen cultures. None of these factors were detected in control wound beds. Monoclonal antibodies against collagen IV and tenascin showed that both were of murine origin. We propose that the frozen cultures of human keratinocytes promote faster reepithelialization through the release of growth factors such as TGF-alpha which directly enhance migration and proliferation of murine keratinocytes, and through the stimulation of murine subepithelial cells, by TGF-beta, to secrete basement membrane proteins such as collagen IV, laminin, and tenascin, which provide a provisional substrate that improves migration of the murine epidermal cells.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Substâncias de Crescimento/biossíntese , Substâncias de Crescimento/farmacologia , Cicatrização/fisiologia , Animais , Células Cultivadas , Colágeno Tipo IV/metabolismo , Células Epidérmicas , Fibronectinas/metabolismo , Congelamento , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Tenascina/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Objetivo: Observar y describir, mediante microscopía confocal, los cambios asociados al proceso de cicatrización de quemadura por álcali, en córneas sanas de conejo. Método: Se realizó microscopía confocal (ConfoScan 2.0, Fortune Technologies Srl.,Italy) para el análisis morfológico del proceso de cicatrización corneal secundaria a quemadura por álcali, previo a la quemadura, a la semana, al mes y a los tres meses. Resultados: Se observó una cicatriz subepitelial cuya densidad aumenta al profundizar al estroma anterior, desde la primer semana hasta 3 meses después. El epitelio se encontró re-epitelizado desde la primer semana a la quemadura por álcali. Conclusiones: La microscopía confocal demuestra la pronta recuperación del epitelio corneal de los conejos, no obstante se requiere de un estudio a mayor largo plazo para determinar si existen cambios en el proceso de cicatrización del estroma corneal.