RESUMO
During the ovarian ontogeny in birds, five fundamental events can be recognized: migration and colonization of the primordial germ cells, differentiation and proliferation of oogonies, an organization of germinal nests, beginning of the meiotic process and folliculogenesis. The knowledge of these events is fundamental for the interpretation of the processes involved in the differentiation of female gametes. However, there are only references for some model species such as Gallus gallus domesticus and Coturnix coturnix. In a previous study, the histological structure of embryonic ovaries of Columba livia was revealed. Therefore, the objective of this work is to characterize the processes of meiosis and folliculogenesis C. livia from the analysis of the expression of the GnRH receptor, the 3ßHSD enzyme and the cell proliferation protein PCNA in embryonic and postnatal ovaries. Therefore, the expression of GnRHR, 3ßHSD, and PCNA was revealed in histological testicular and ovarian preparations in embryos (stages 41-43) and neonates (2, 5, 7, 10 and 75â¯days post-hatching). The present study demonstrates that the fate of germline cells is dictated by their location during gonadal development. Thus, the germline cells located in the cortex of the left gonad enter meiosis, while those in the right gonad and those in the medulla of the left ovary fail to go into meiosis. This indicates that somatic signals, instead of an autonomous cellular mechanism, regulate the entry of the germline cells into meiosis in the C. livia embryo. Future studies will be focused on the analysis of proteins associated with meiotic events and folliculogenesis in embryonic and neonatal ovaries of C. livia, to evaluate the regulation of meiosis in vitro.
Assuntos
3-Hidroxiesteroide Desidrogenases/metabolismo , Columbidae/metabolismo , Meiose , Folículo Ovariano/crescimento & desenvolvimento , Receptores LHRH/metabolismo , Animais , Proliferação de Células , Columbidae/embriologia , Embrião não Mamífero/citologia , Feminino , Células Germinativas/metabolismo , Imuno-Histoquímica , Oócitos/citologia , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
The spatial organization of cells during tissue differentiation is a crucial process in the morphogenesis of vertebrates. This process involves the movement, separation, and connection of cells. It is essential to elucidate the molecular mechanisms involved in these processes for the understanding of animal morphogenesis. Cell-cell adhesion molecules, called cadherins, are involved in the selective adhesion of cells. In the case of birds, the expression of these molecules in various organ systems during embryonic development has been reported in Gallus gallus domesticus. In this work, we present the immunohistochemical analysis of the differential expression of E and N-cadherin binding molecules in Columba livia embryos at various stages of gonadal morphogenesis. The expression of E and N-cadherin in embryos corresponding to the stages 41, 43 and in neonates of 2, 5, 7 and 75 post-hatching days were assessed by immunohistochemistry. Results revealed the expression of N-cadherin in the plasma membrane and the perinuclear zone of germline cells in ovaries and testes. However, the expression of E-cadherin was noticed with similar immunoreactivity pattern, in Sertoli cells and in the cells of the follicular nests. The differential expression of follicular cells and Sertoli cells positive for E-cadherin and germline cell N-cadherin positive cells were evidenced in the present work at the cell-cell interaction level. Future studies will focus on determining the expression of E and N-cadherin molecules during the migration of the primordial germ cells and the colonization of the genital ridge.
Assuntos
Caderinas/metabolismo , Galinhas/metabolismo , Morfogênese/fisiologia , Testículo/metabolismo , Animais , Columbiformes , Feminino , Imuno-Histoquímica/métodos , Masculino , Ovário/metabolismo , Células de Sertoli/citologiaRESUMO
In this work, testicular ontogeny is analyzed at the anatomical, histological and immunohistochemical levels; the latter through the detection of GnRHR and PCNA in the testicles of embryos, neonates and juveniles of Columba livia. We analyzed 150 embryos, 25 neonates and 5 juveniles by means of observations under a stereoscopic magnifying glass and scanning electron microscope (SEM). The histological analysis was performed using hematoxylin-eosin staining techniques and the PAS reaction. For the immunohistochemical analysis, the expression of GnRHR and PCNA in embryos corresponding to stages 41, 43 and in neonates of 2, 5, 7 and 75 days post-hatch was revealed in testicular histological preparations. That gonadal outline is evident in stage 18. In stage 29, the testes are constituted of a medulla in which the PGCs are surrounded by the Sertoli cells, constituting the seminiferous tubules. From stage 37 a greater organization of the tubules is visualized and at the time of hatching the testicle is constituted of the closed seminiferous tubules, formed of the PGCs and Sertoli cells. The Leydig cells are evident outside the tubules. In the juvenile stages, the differentiation of germline cells and the organization of small vessels that irrigate the developing testicle begin to be visible. In the analyzed stages, the immunodetection of the GnRHR receptor and PCNA revealed specific marking in the plasma membrane and in the perinuclear zone for GnRHR and in the nucleus of the germline cells in juvenile testicles for PCNA. These results can be used as a basis for further study of endocrine regulation events during testicular ontogeny in avian species.
Assuntos
Receptores LHRH/metabolismo , Testículo/crescimento & desenvolvimento , Animais , Columbiformes , Imuno-Histoquímica , Masculino , Antígeno Nuclear de Célula em Proliferação/metabolismo , Testículo/anatomia & histologia , Testículo/embriologiaRESUMO
El objetivo del presente estudio fue optimizar la implementación de cultivos primarios a partir de muestras de carcinoma renal de células claras (CRCC) para comprobar la conservación del fenotipo lipogénico contra cortes fijados del mismo origen. Se utilizaron muestras de pacientes con CRCC, evaluándose diversas metodologías y condiciones experimentales de digestión de muestras, adherencia y despegue celular, fenotipo lipogénico, potencial de clonación, proliferación y capacidad de migración. El mayor rendimiento y viabilidad celular se verificó mediante digestión con colagenasa. La adherencia inicial se logró a las 24 hs de incubación, utilizando placas plásticas de cultivo, recubiertas con colágeno comercial y gelatina 0,2% en la mayoría de las muestras analizadas (60% de los casos). Se obtuvieron monocapas, con potencial de migración, en un 40% de los casos, tras 5 ± 1 días de incubación. El promedio de subcultivos fue de 3 ± 1. Este estudio permitió estandarizar cultivos primarios de CRCC comprobándose la conservación de la fenotipia lipogénica, logrando de dicha manera una herramienta importante y útil para el estudio de la biología tumoral y el ensayo de nuevas terapéuticas
The aim of this study was to optimize the implementation of primary cultures from samples of renal clear cell carcinoma (CRCC) to check the conservation of the lipogenic phenotype. CRCC Patient samples were used, in order to evaluate different methodologies and the experimental conditions of sample digestion, cell adhesion and lipogenic phenotype, proliferation and migration ability. The highest yield in cell number and viability was assessed using collagenase digestion. The initial adhesion was achieved after 24 hours of incubation in plastic plates recoverd with commercial collagen or 0.2% gelatin (60% of cases). Monolayers, with migration potential, were obtained in 40% of all cases, after 5 ± 1 days of incubation. The subcultures average was 3 ± 1. This study allowed us to standardize primary cultures of CRCC and check the conservation of the lipogenic phenotyping, achieving in this way an important and useful tool to study the tumor biology.
O objetivo deste trabalho foi otimizar a implementação de culturas primárias de amostras de carcinoma de células claras renal (CRCC) para verificar conservação fenótipo lipogenic contra os cortes previstos a mesma origem. As amostras dos pacientes foram utilizados CRCC, avaliando diferentes metodologias e as condições experimentais da digestão de amostras, adesão celular e fenótipo clonagem potencial take-lipogenic, proliferação e capacidade de migração. O maior rendimento e a viabilidade celular foi avaliada por digestão com colagenase. A adesão inicial foi obtida após 24 horas de incubação com colagénio e gelatina comercial 0,2% em 60% dos casos. As monocamadas foram obtidos em 40% após 5 ± 1 dias de incubação com o potencial de migração. As subculturas média foi de 3 ± 1. Este estudo nos permitiu padronizar culturas primárias de CRCC são verificados quanto à conservação da fenotipagem lipogenic, conseguindo desta forma um importante e útil para o estudo da biologia do tumor e teste de nova ferramenta terapêutica
Assuntos
Humanos , Carcinoma de Células Renais/diagnóstico , Neoplasias Renais/diagnóstico , Técnicas de Cultura/métodos , Cultura Primária de Células/métodosRESUMO
Due to variability of venom components from the same species of snakes that inhabit different regions, particular properties of the venom of Crotalus durissus terrificus that inhabits the North-East of Argentina were studied. Gyroxin, a thrombin-like enzyme, was isolated from this venom by gel filtration and affinity chromatography, it was found to be homogeneous according to SDS-PAGE, with a molecular weight of 33 kDa. [quot ]Gyroxin syndrome[quot ] in mice was tested and it showed changes in the animal behavior, confirming that the isolated thrombin-like enzyme is gyroxin. Effects of this enzyme and the crude venom on mice plasmatic fibrinogen levels were determined. The mice plasma fibrinogen decreased rapidly until incoagulability during the first hour after thrombin-like enzyme injection, then reaching its normal level 10 hours after injection; whereas crude venom resulted in a 60% decrease of the mice plasma fibrinogen, reaching its normal level after the same period of time. After 1 hour of gyroxin inoculation, intravascular coagulation was observed in histological cuttings of lung, cardiac muscle and liver. The isolated enzyme showed strong hydrolyzing activity on fibrinogen and fibrin in vitro, whereas the crude venom exhibited weak hydrolyzing activity on both substrates. It is probable that this very low activity is due to the low percentage of the enzyme in the crude venom. Decreasing of plasmatic fibrinogen levels may be due to either the coagulant or hydrolyzing actions of the enzyme.
Teniendo en cuenta la variabilidadde los componentes del veneno de serpientes de una misma especie que habitan regiones diferentes, se decidióestudiar las propiedades particulares del veneno de Crotalus durissus terrificus que habita el nordeste de Argentina, Giroxina, una enzima con actividad trombínica, fue aislada del veneno por cromatografía de filtración por gel y de afinidad; se comprobó su homogeneidad y se determinó su peso molecular, 33 kDa, por SDSPAGE. Se ensayó el síndrome giroxina en ratones, los que mostraron cambios en el comportamiento, confirmandoque la enzima tipo trombina aislada es giroxina. Se evaluó la acción de esta enzima sobre los niveles de fibrinógeno plasmático en ratones, comparándola con la del veneno crudo. Se comprobó que la enzima provoca una disminución de los niveles plasmáticos de fibrinógeno hasta la incoagulabilidad, durante la primer hora de inoculación, mientras que el veneno entero produjo una reducción del nivel plasmático en un 60%; sin embargo, en ambos casos, se evidenció una rápida reposición de fibrinógeno plasmático, alcanzando sus valores normales en un plazo de 10 horas. Se observó coagulación intravascular con la administración de giroxina una hora después de la inoculación, evidenciados en estudios histológicos de tejido pulmonar, cardíaco y hepático. En ensayos realizados in vitro, la enzima aislada mostró capacidad de degradar fibrinógeno como así también coágulos de fibrina, mientras que el veneno exhibió débil actividad hidrolítica sobre ambos sustratos. Es probable que esta baja actividad sea debida a la baja concentración de la enzima en el veneno. La disminución de los niveles de fibrinógeno plasmático observado en ratones se debería a la acción coagulante de la enzima, sin embargo no se descarta que también contribuya a este proceso una acción hidrolítica sobrefibrinógeno y fibrina por parte de la enzima.
Assuntos
Animais , Feminino , Masculino , Camundongos , Crotalus , Fibrinogênio/metabolismo , Trombina/metabolismo , Venenos de Crotalídeos/enzimologia , Argentina , Coagulantes/farmacologia , Fígado/patologia , Pulmão/patologia , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/metabolismo , Venenos de Crotalídeos/farmacologiaRESUMO
Paclitaxel (Px) is a cancer chemotherapeutic agent that causes bone marrow (BM) cytotoxicity by microtubule stabilization and by modifications in the expression of several genes. Hematopoietic progenitors show severe alterations following Px injury. Erythropoietic recovery should be accompanied by changes in the expression of transcription factors such as c-MYB, GATA-1, NF-E2, Bcl-x(L), and erythropoietin receptor (Epo-R). The aim of this work was to study the in vivo recovery of erythropoiesis and to correlate transcription factors, Bcl-x(L), and Epo-R expressions to apoptosis and changes in proliferation of murine erythroid progenitors following a single dose of Px (29 mg/kg, i.p.). BM total and differential cellularities, apoptosis (TdT-mediated dUTP Nick-End Labeling [TUNEL] assay), clonogenic assays, and immunoblots for transcription factors, Epo-R, and Bcl-x(L) were performed each day for 5 days post-injury. Apoptosis (24 +/- 0.81%, P < 0.01), inhibition of colony growth (burst-forming units-erythroid [BFU-E] and granulocyte-erythroid-macrophage [GEM]), and decrease in BM cellularities (28 +/- 4.2% of control) were maximal at 24 h following Px. The highest apoptosis was concomitant with the lowest BM cellularities. Apoptosis returned to normal values (3.08 +/- 0.61%) by day 3 post-Px. Up-regulation of c-MYB, GATA-1, Epo-R, and Bcl-x(L) expressions were observed between 24 and 48 h following Px. Correlations among c-MYB, GATA-1, Bcl-x(L), and Epo-R were extremely significant. Maximal expression of NF-E2 was observed on day 3 concomitant with the rise (threefold) of early erythroid precursors (BFU-E). Thus, cells that survive injury seem to be stimulated to produce early (24-48 h) erythroid-related and antiapoptotic proteins. Therefore, the results suggest an in vivo interplay between specific transcription factors and Bcl-x(L) during progenitor cell survival and proliferation; mechanisms triggered to restore size and composition of the erythroid compartment.
Assuntos
Antineoplásicos Fitogênicos/toxicidade , Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eritropoese/efeitos dos fármacos , Genes myb/genética , Paclitaxel/toxicidade , Receptores da Eritropoetina/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Sobrevivência Celular , Ensaio de Unidades Formadoras de Colônias , Células Precursoras Eritroides/efeitos dos fármacos , Fatores de Ligação de DNA Eritroide Específicos , Feminino , Fator de Transcrição GATA1 , Expressão Gênica/efeitos dos fármacos , Genes bcl-1/genética , Processamento de Imagem Assistida por Computador , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Cinética , Camundongos , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Receptores da Eritropoetina/biossínteseRESUMO
Due to variability of venom components from the same species of snakes that inhabit different regions, particular properties of the venom of Crotalus durissus terrificus that inhabits the North-East of Argentina were studied. Gyroxin, a thrombin-like enzyme, was isolated from this venom by gel filtration and affinity chromatography, it was found to be homogeneous according to SDS-PAGE, with a molecular weight of 33 kDa. [quot ]Gyroxin syndrome[quot ] in mice was tested and it showed changes in the animal behavior, confirming that the isolated thrombin-like enzyme is gyroxin. Effects of this enzyme and the crude venom on mice plasmatic fibrinogen levels were determined. The mice plasma fibrinogen decreased rapidly until incoagulability during the first hour after thrombin-like enzyme injection, then reaching its normal level 10 hours after injection; whereas crude venom resulted in a 60% decrease of the mice plasma fibrinogen, reaching its normal level after the same period of time. After 1 hour of gyroxin inoculation, intravascular coagulation was observed in histological cuttings of lung, cardiac muscle and liver. The isolated enzyme showed strong hydrolyzing activity on fibrinogen and fibrin in vitro, whereas the crude venom exhibited weak hydrolyzing activity on both substrates. It is probable that this very low activity is due to the low percentage of the enzyme in the crude venom. Decreasing of plasmatic fibrinogen levels may be due to either the coagulant or hydrolyzing actions of the enzyme.(AU)
Teniendo en cuenta la variabilidadde los componentes del veneno de serpientes de una misma especie que habitan regiones diferentes, se decidióestudiar las propiedades particulares del veneno de Crotalus durissus terrificus que habita el nordeste de Argentina, Giroxina, una enzima con actividad trombínica, fue aislada del veneno por cromatografía de filtración por gel y de afinidad; se comprobó su homogeneidad y se determinó su peso molecular, 33 kDa, por SDSPAGE. Se ensayó el síndrome giroxina en ratones, los que mostraron cambios en el comportamiento, confirmandoque la enzima tipo trombina aislada es giroxina. Se evaluó la acción de esta enzima sobre los niveles de fibrinógeno plasmático en ratones, comparándola con la del veneno crudo. Se comprobó que la enzima provoca una disminución de los niveles plasmáticos de fibrinógeno hasta la incoagulabilidad, durante la primer hora de inoculación, mientras que el veneno entero produjo una reducción del nivel plasmático en un 60%; sin embargo, en ambos casos, se evidenció una rápida reposición de fibrinógeno plasmático, alcanzando sus valores normales en un plazo de 10 horas. Se observó coagulación intravascular con la administración de giroxina una hora después de la inoculación, evidenciados en estudios histológicos de tejido pulmonar, cardíaco y hepático. En ensayos realizados in vitro, la enzima aislada mostró capacidad de degradar fibrinógeno como así también coágulos de fibrina, mientras que el veneno exhibió débil actividad hidrolítica sobre ambos sustratos. Es probable que esta baja actividad sea debida a la baja concentración de la enzima en el veneno. La disminución de los niveles de fibrinógeno plasmático observado en ratones se debería a la acción coagulante de la enzima, sin embargo no se descarta que también contribuya a este proceso una acción hidrolítica sobrefibrinógeno y fibrina por parte de la enzima. (AU)
Assuntos
Animais , Feminino , Masculino , Camundongos , Venenos de Crotalídeos/enzimologia , Crotalus , Fibrinogênio/metabolismo , Trombina/metabolismo , Argentina , Coagulantes/farmacologia , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/metabolismo , Venenos de Crotalídeos/farmacologia , Fígado/patologia , Pulmão/patologiaRESUMO
Paclitaxel is a drug widely used in several oncological trials. Like other antineoplastics, it causes severe neutropenia. However, its effects on erythropoiesis are not as well known. This study analyzes the recovery of normal murine hematopoiesis after single dose paclitaxel administration (29 mg/kg i.p.) over 20 days. Different assays were used to analyze the restorative kinetics from primitive early progenitors to mature functional erythroid cells. Proliferation of the erythroid compartment was assessed by DNA microculture assays in medullar and splenic cells stimulated with recombinant human erythropoietin (rh Epo 0-500 mU/ml). Enhancement of early hematopoietic progenitors was determined using clonogenic assays and erythroid terminal maturation by 59Fe incorporation. Peripheral hematologic parameters and cellularities in both tissues were also determined on each day of the experimental schedule. At 2 days post-paclitaxel treatment, medullar cellularity diminished drastically (> 90%) and 59Fe incorporation decreased in all compartments. DNA assay revealed maximum sensitivity to Epo (p < 0.05 with 15 mU/ml) while clonogenic cultures failed to show significant results. At 5 days both bone marrow and spleen semisolid cultures showed great expansion of early hematopoietic progenitors (about 5- and 83-fold. respectively). Hormonal sensitivity decreased progressively along the experiment. Splenic cultures showed a linear dose-response to rh Epo at day 5 post-paclitaxel administration (p < 0.05 with 125 mU/ml). Medullar and splenic total progenitor colony-forming units (CFU) scorings with and without rh Epo revealed a notable enhancement at 5 days post-paclitaxel treatment. Data from this study suggest that paclitaxel causes deep injury in the erythropoietic compartment, including temporary variations of Epo sensitivity in late bone marrow erythroid progenitors, early multilineage hematopoietic explosion and terminal erythroid precursors depletion as a result of a complex microenvironmental restitutive regulation.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Células Precursoras Eritroides/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Paclitaxel/farmacologia , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/sangue , Divisão Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Relação Dose-Resposta a Droga , Eritropoetina/farmacologia , Feminino , Radioisótopos de Ferro/metabolismo , Camundongos , Células Mieloides/efeitos dos fármacos , Paclitaxel/administração & dosagem , Paclitaxel/sangue , Proteínas Recombinantes , Baço/citologia , Baço/efeitos dos fármacosRESUMO
Hypoxia is the best physiological stress to disturb the erythropoietic steady state. The present study has been undertaken in the aim to analize the splenic erythropoietic proliferative response with different doses of recombinant human erythropoietin under hypoxic conditions along 18 days using the DNA synthesis assay. Normoxic splenic progenitors failed to show significative erythroid replication at 0 days. A clearly rh Epo response was noticed from 2 to 8 days of hypoxia. Splenic proliferation returned to basal pattern from 10 days to the end of the experience. The highest proliferative activity, 25 fold increase over control (p < 0.001), was found at 6 days from 62.5 to 250 mU/ml rh Epo. These results support suggestions that hypoxia induces a transiently erythroid splenic proliferative response changing its quantitative parameters in the Epo dose-response relationship during the physiological adaptation.
Assuntos
Eritropoese/fisiologia , Eritropoetina/administração & dosagem , Hipóxia/fisiopatologia , Baço/patologia , Animais , Técnicas de Cultura de Células , Divisão Celular , DNA/biossíntese , Células Precursoras Eritroides/fisiologia , Feminino , Hipóxia/patologia , Camundongos , Camundongos Endogâmicos , Fatores de TempoRESUMO
La hipoxia es un estímulo que perturba el equilibrio hemopoyético, particularmente el compartimiento eritroide. Ante esta situación el sistema se acomoda para asimilar las variaciones del medio estableciendo un nuevo estado de equilibrio. Este trabajo se diseñó para analizar la variación de sensibilidad de las células esplénicas a distintas concentraciones de eritropoyetina humana recombinante (rh Epo), sometiendo al animal donante a diferentes tiempos de hipoxia y midiendo la respuesta proliferativa por la incorporación de timidina triatiada (3-HdTR). Los progenitores esplénicos muestran índices de estimulación crecientes en función de la hipoxia con un máximo entre 6 y 8 días. A partir de allí desciende la capacidad proliferativa eritroide del bazo. La actividad máxima se verifica con 32,5 y 62,5 rh-Epo mU/ml. La hipoxia cambia el comportamiento eritroideo esplénico con una actividad máxima entre los 6 y 8 días, expresado entre otras variables por un aumento de la proliferación de los progenitores eritroides hasta 25 veces los valores control. Estos resultados sugieren que bajo strees hemopoyético (hipoxia) las células esplénicas exhiben un incremento de sensibilidad a la rh Epo en forma transitoria
Assuntos
Humanos , Animais , Ratos , Eritropoetina , Hematopoese Extramedular/efeitos dos fármacos , Hipóxia/fisiopatologia , Baço/efeitos dos fármacosRESUMO
La hipoxia constituye el mejor stress fisiológico para la pertubación del estado estacionario eritropoyético. El present estudio tiende a analizar la respuesta proliferativa eritropoyética esplénica con diferentes dosis de eritropoyentina humana recombinante bajo condiciones hipóxicas a lo largo 18 días mediante el ensayo de síntesis del DNA. Los progenitores esplénicos normóxicos no sufren proliferación eritroide significativa al día 0. Una clara respuesta proliferativa a rh Epo se verificó entre los 2 y 8 días de hipoxia. La proliferación de los progenitores eritroides esplénicos hipóxicos retornó a un patrón basal desde los 10 días hasta el final de la experiencia. La mayor creatividad proliferativa, 25 veces sobre el control (p<0.001), se produjo a los 6 días de condicionamiento desde 62.5 hasta 250mU/ml de rh Epo. estos resultados son concordantes con el concepto que durante la daptación fisiológica a la hipoxia, las células progenitoras eritroides esplénicas modifican transitoriamente su tasa proliferativa observable por variaciones en la relación dosis-respueta a Epo
Assuntos
Feminino , Camundongos , Animais , Baço/citologia , Eritropoese/fisiologia , Eritropoetina/administração & dosagem , Hipóxia/fisiopatologia , Adaptação Fisiológica , Camundongos Endogâmicos , Técnicas de Cultura de Células , Células Precursoras Eritroides/fisiologia , DNA/biossíntese , Fatores de TempoRESUMO
Hypoxia is the best physiological stress to disturb the erythropoietic steady state. The present study has been undertaken in the aim to analize the splenic erythropoietic proliferative response with different doses of recombinant human erythropoietin under hypoxic conditions along 18 days using the DNA synthesis assay. Normoxic splenic progenitors failed to show significative erythroid replication at 0 days. A clearly rh Epo response was noticed from 2 to 8 days of hypoxia. Splenic proliferation returned to basal pattern from 10 days to the end of the experience. The highest proliferative activity, 25 fold increase over control (p < 0.001), was found at 6 days from 62.5 to 250 mU/ml rh Epo. These results support suggestions that hypoxia induces a transiently erythroid splenic proliferative response changing its quantitative parameters in the Epo dose-response relationship during the physiological adaptation.
RESUMO
La hipoxia constituye el mejor stress fisiológico para la pertubación del estado estacionario eritropoyético. El present estudio tiende a analizar la respuesta proliferativa eritropoyética esplénica con diferentes dosis de eritropoyentina humana recombinante bajo condiciones hipóxicas a lo largo 18 días mediante el ensayo de síntesis del DNA. Los progenitores esplénicos normóxicos no sufren proliferación eritroide significativa al día 0. Una clara respuesta proliferativa a rh Epo se verificó entre los 2 y 8 días de hipoxia. La proliferación de los progenitores eritroides esplénicos hipóxicos retornó a un patrón basal desde los 10 días hasta el final de la experiencia. La mayor creatividad proliferativa, 25 veces sobre el control (p<0.001), se produjo a los 6 días de condicionamiento desde 62.5 hasta 250mU/ml de rh Epo. estos resultados son concordantes con el concepto que durante la daptación fisiológica a la hipoxia, las células progenitoras eritroides esplénicas modifican transitoriamente su tasa proliferativa observable por variaciones en la relación dosis-respueta a Epo (AU)
Assuntos
Feminino , Camundongos , Animais , Hipóxia/fisiopatologia , Eritropoese/fisiologia , Eritropoetina/administração & dosagem , Baço/citologia , Células Precursoras Eritroides/fisiologia , Adaptação Fisiológica , DNA/biossíntese , Técnicas de Cultura de Células , Fatores de Tempo , Camundongos EndogâmicosRESUMO
La hipoxia es un estímulo que perturba el equilibrio hemopoyético, particularmente el compartimiento eritroide. Ante esta situación el sistema se acomoda para asimilar las variaciones del medio estableciendo un nuevo estado de equilibrio. Este trabajo se diseñó para analizar la variación de sensibilidad de las células esplénicas a distintas concentraciones de eritropoyetina humana recombinante (rh Epo), sometiendo al animal donante a diferentes tiempos de hipoxia y midiendo la respuesta proliferativa por la incorporación de timidina triatiada (3-HdTR). Los progenitores esplénicos muestran índices de estimulación crecientes en función de la hipoxia con un máximo entre 6 y 8 días. A partir de allí desciende la capacidad proliferativa eritroide del bazo. La actividad máxima se verifica con 32,5 y 62,5 rh-Epo mU/ml. La hipoxia cambia el comportamiento eritroideo esplénico con una actividad máxima entre los 6 y 8 días, expresado entre otras variables por un aumento de la proliferación de los progenitores eritroides hasta 25 veces los valores control. Estos resultados sugieren que bajo strees hemopoyético (hipoxia) las células esplénicas exhiben un incremento de sensibilidad a la rh Epo en forma transitoria (AU)
Assuntos
Humanos , Animais , Ratos , Eritropoetina , Hematopoese Extramedular/efeitos dos fármacos , Hipóxia/fisiopatologia , Baço/efeitos dos fármacosRESUMO
Representative specimens from two classes of Vertebrata Sub-Phyllum, Bufo paracnemis (amphibian) and Gallus domesticus (avian) were made anemic by phenylhydrazine treatment. Appearance of serum factors able to stimulate the proliferation of mammalian erythroid cells was tested. Normal and anemic sera from Gallus domesticus and Bufo paracnemis were fractioned by alcoholic treatment and assayed by the post-hypoxic mice method, showing null uptake of 59Fe. When assayed in semisolid cultures using bone marrow murine cells at different times of incubation (CFU-E and BFU-E colonies), anemia Gallus domesticus serum showed high stimulatory activity, while anemic Bufo paracnemis serum was unable to enhance erythroid proliferation. Gel filtration chromatography of partially purified avian samples on Sephadex G-150 showed three molecular entities responsible for biological activity in vitro, with an apparent molecular weight of 29, 14 and 10 KD respectively. They were submitted to several treatments and then tested for biological activity. All factors were heat stable, sensitive to neuraminidase treatment, while dithiothreitol caused loss of activity on low molecular weight proteins. These results suggest at least under these experimental conditions, the presence of analogous erythroid factors among homeotherms amniotas.
Assuntos
Anemia/sangue , Sangue , Fatores Estimuladores de Colônias/sangue , Células Precursoras Eritroides/efeitos dos fármacos , Animais , Células da Medula Óssea , Bufonidae , Cromatografia em Gel , Feminino , Masculino , Camundongos , Aves DomésticasRESUMO
Representative specimens from two classes of Vertebrata Sub-Phyllum, Bufo paracnemis (amphibian) and Gallus domesticus (avian) were made anemic by phenylhydrazine treatment. Appearance of serum factors able to stimulate the proliferation of mammalian erythroid cells was tested. Normal and anemic sera from Gallus domesticus and Bufo paracnemis were fractioned by alcoholic treatment and assayed by the post-hypoxic mice method, showing null uptake of 59Fe. When assayed in semisolid cultures using bone marrow murine cells at different times of incubation (CFU-E and BFU-E colonies), anemia Gallus domesticus serum showed high stimulatory activity, while anemic Bufo paracnemis serum was unable to enhance erythroid proliferation. Gel filtration chromatography of partially purified avian samples on Sephadex G-150 showed three molecular entities responsible for biological activity in vitro, with an apparent molecular weight of 29, 14 and 10 KD respectively. They were submitted to several treatments and then tested for biological activity. All factors were heat stable, sensitive to neuraminidase treatment, while dithiothreitol caused loss of activity on low molecular weight proteins. These results suggest at least under these experimental conditions, the presence of analogous erythroid factors among homeotherms amniotas.