RESUMO
Los modelos experimentales en rata han sido de gran utilidad en las evaluaciones terapéuticas o de reemplazo de células en enfermedades neurodegenerativas. Se ha comprobado que las células de la médula ósea (CMO) de ratas pueden diferenciarse en células que no forman parte de sus linajes normales. Hay evidencias de estos procesos de trans-diferenciación, pero aún no se conocen los mecanismos moleculares que activan estos procesos. El propósito de nuestro trabajo fue estudiar el polimorfismo genético del ADN de los tipos celulares que conforman las CMO y las células del sistema nervioso central (SNC), estríatales y de la corteza de ratas mediante la técnica de RAPD. Las CMO, las células mononucleares (CMMO), las células estromales (CEMO) y las del SNC fueron obtenidas de ratas, y su ADN genómico fue purificado y amplificado mediante la técnica de RAPD, utilizando 15 cebadores al azar. Se construyó un dendograma de las bandas de amplificación generadas utilizando el método de UPGMA. Las células estudiadas según el análisis del RAPD quedaron en 2 grupos bien definidos, pudiéndose diferenciar las CEMO del resto de las células estudiadas. Los cebadores OPA-6, 7 y 12, mostraron el polimorfismo genético de los linajes de células estudiadas. Mediante la técnica de RAPD se demostró la variabilidad genética entre las CEMO y las CMMO, células estriadas y de corteza que mostraron una homogeneidad genética, proponiéndose marcadores específicos de RAPD para cada grupo de células. Este es el primer estudio del polimorfismo genético de las CMO y del SNC de ratas.
Experimental models have been of grate usefulness in the therapeutic or replacement cells in neurodegenerative diseases. It has been demonstrated that bone marrow cells (BMC), can be difefferentiated in cells that do not form part of their normal lineage. There is evidence of these trans-differentiation processes in these cells, but nevertheless, molecular mechanisms that activate these differentiation process still not known. The purpose of our work was to study the genetic polymorphism of those cellular types; that conform the rat bone marrow cells (BMC) as well as those of the central nervous system (CNS), striatum cells and cortex ones, trough RAPD technique. BM, mononuclear cells (BMMC), estromal cells (BMSC) and the CNS cells were obtained from rats and genomic ADN was purified and amplified through RAPD technique, using 15 random primers. A dendogram was constructed according to UPGMA method of the amplifying RAPD bands. Studied cells as- according to the RAPD analysis- were grouped into 2 well- defined groups, as CEMO coud be differentiated from the rest of studied cells. OPA-6, 7 and 12 primers showed the genetic polymorphism of the studied lineages cells. Also will be proposed specific RAPD genetic markers. Through RAPD technique permitted the genetic variability was demonstrated betwen BMEC and BMMC of striated cells and of cortex, which demonstratd a genetic homogeneity through RAPD technique so specific genetic markers of RAPD were thus propose for each group of cells. These constitute the first study on genetic polymorphism of BMC and CNS.
Assuntos
Medula Óssea/anormalidades , Medula Óssea/crescimento & desenvolvimento , Medula Óssea/imunologia , Medula Óssea/ultraestrutura , Polimorfismo Genético/fisiologia , Polimorfismo Genético/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Sistema Nervoso Central/anormalidades , Sistema Nervoso Central/lesões , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/microbiologia , Sistema Nervoso Central/ultraestruturaRESUMO
BACKGROUND: Bone marrow-derived stem cell transplantation is a potentially viable therapeutic option for the treatment of neurodegenerative disease. MATERIAL/METHODS: We have isolated bone marrow stem cells by standard method. We then evaluated the survival of rats' bone marrow mononuclear cells implanted in rats' brain. The cells were extracted from rats' femurs, and marked for monitoring purposes by adenoviral transduction with Green Fluorescent Protein (GFP). Labeled cells were implanted within the area of rats' striatum lesions that were induced a month earlier employing quinolinic acid-based method. The implants were phenotyped by monitoring CD34; CD38; CD45 and CD90 expression. Bone marrow stromal cells were extracted from rats' femurs and cultivated until monolayer bone marrow stromal cells were obtained. The ability of bone marrow stromal cells to express NGF and GDNF was evaluated by RT-PCR. RESULTS: Implanted cells survived for at least one month after transplantation and dispersed from the area of injection towards corpus callosum and brain cortex. Interestingly, passaged rat bone marrow stromal cells expressed NGF and GDNF mRNA. CONCLUSIONS: The bone marrow cells could be successfully transplanted to the brain either for the purpose of trans-differentiation, or for the expression of desired growth factors.
Assuntos
Células da Medula Óssea/citologia , Transplante de Tecido Encefálico , Encéfalo/patologia , Sobrevivência de Enxerto , Neurônios/metabolismo , Transplante de Células-Tronco , Adenoviridae , Animais , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Regulação da Expressão Gênica , Leucócitos Mononucleares/virologia , Masculino , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Ácido Quinolínico , Ratos , Ratos Sprague-Dawley , Coloração e Rotulagem , Fatores de TempoRESUMO
Impairments in motor coordination and cognition in normal and pathological aging are often accompanied by structural changes, that is, loss of synapses and neurons. Also, it has been shown recently that bone marrow stem cells can give origin to cells of different tissues, including neural cells. Given the therapeutic implications of increasing health and functional possibilities in the aged brain, we have tested the effects of rat femur bone marrow stem cells (rBMSCs) grafting to the striatum hippocampus of aged rats with motor or cognitive deficits, respectively. Bone marrow cells were transduced with an adenovirus driving the expression of green fluorescence protein (GFP) and other classic stains to determine their migration, engraftment, differentiation, and associated behavioral recovery. Five weeks after it, control and grafted rats were re-evaluated with the Morris Water Maze test, Passive avoidance, open-field, motor coordination, and Marshall tests and perfused. Brains were processed and analyzed for fluorescent protein expression. GFP was detected in cells with some differentiation degree into neural-like cells. Their exact phenotype is yet to be determined. A significant functional recovery was observed 6 weeks after grafting, suggesting a trophic interaction between rBMSCs and the aged/dystrophic host brain, or with the host brain progenitor cells and/or by increasing the number of functional cells at striatum or hippocampus, suggesting that the aging brain keeps its functional plasticity as well as that BMSCs are interesting candidates for cell replacement therapies in neurodegenerative disorders.
Assuntos
Envelhecimento , Células da Medula Óssea/citologia , Transplante de Medula Óssea , Corpo Estriado/patologia , Hipocampo/patologia , Memória , Células-Tronco/citologia , Animais , Diferenciação Celular , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Masculino , Aprendizagem em Labirinto , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Bone marrow stromal cells (BMSC) have attracted interest through their possible use for cell therapy in neurological diseases. Recent reports demonstrated that these cells are able to migrate and have potential for neuronal differentiation after transplantation into brain parenchyma. The objective of this work was determine whether rat BMSC express NGF and GDNF, in order to study its potential application for treatment of neurodegenerative diseases. BMSC were harvested from male rats and cultured in DMEM supplemented with 20% fetal bovine serum. At passage 6 the total RNA was isolated using TriZol reactive. RT-PCRs to evaluate the expression of NGF and GDNF using specific primers were carried out. Our results indicate that rat BMSC have potential to produce NGF and GDNF. We have not found any report in favor of GDNF or NGF production from rat BMSC.
Assuntos
Células da Medula Óssea/metabolismo , Fator de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/biossíntese , Células Estromais/metabolismo , Animais , Células Cultivadas , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Guanidinas/farmacologia , Masculino , Fenóis/farmacologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Soluções/farmacologia , Temperatura , Fatores de TempoRESUMO
A longitudinal study was performed with sera and urine of patients with acquired immune deficiency syndrome (AIDS), taken before, during and after clinically Toxoplasma infection. The tested patients were followed for an average of two years. The titres of the specific IgG and IgM antibodies were measured by an indirect fluorescent antibody test (IFAT), and the appearance of circulating antigens of T. gondii was determined in 36 urine samples of 13 patients with neurotoxoplasmosis by means of the coagglutination test. The presence of T. gondii antigens in the urine of AIDS patients by this test was correlated with the immunoblot technique, with clinical symptoms and also with pathological findings. Our results indicate that the detection of T. gondii antigens in the urine of AIDS patients can be regarded as a rapid and efficient method for the diagnosis of acute toxoplasmosis.
Assuntos
Humanos , Síndrome da Imunodeficiência Adquirida/parasitologia , Toxoplasma/parasitologia , Toxoplasmose/diagnósticoRESUMO
La Encefalitis Toxoplasmica (ET) es la mas importante complicacion infecciosa del Sistema Nirvioso Central en pacientes de SIDA. Anticuerpos ant-Toxoplasma gondiin fueron detectados en 57 de 79 pacientes de SIDA (71 por cento). De estos seropositivos, desarrollaron la enfermedad (ET) 14 (25 por cento), en los que coincidentemente se detecto la presencia de antigeno del parasito en orina y por tanto fueron objeto de una terapia efectiva, con la subsecuente desaparicion de los sintomas y de los antigenos excretados. Por los resultados del presente trabajo, consideramos lo util de monitorear en estos pacientes la presencia de antigenos de T. gondii con el objetivo de aplicar oportunamente metodos quimoprofilacticos que eviten el surgimiento de manisfestaciones neurologicas en estos pacientes.
Assuntos
Humanos , Masculino , Adulto , Manifestações Neurológicas , Síndrome da Imunodeficiência Adquirida/complicações , Toxoplasmose/diagnóstico , Encefalite/etiologia , Toxoplasmose/imunologiaRESUMO
A simple and rapid staphylococcal coagglutination test for the detection of Toxoplasma gondii antigens in mice urine is described. A suspension of protein-A containing Staphylococcus aureus coated with rabbit hyperimmune serum was used as reagent. The sensitivity of the antigen assay was found to be at least 118 ng of the antigen protein per ml. No coagglutination was observed when the reagent was challenged against antigenic solutions of other parasites. The suitability of the method for detecting antigens of T. gondii in urine samples was studied by experimental toxoplasma infection in mice. Before the staphylococcal test, the urine samples were double serially diluted in 0.1 M PBS. From the second day on all samples from infected mice were positive at 1/16 dilution. At this dilution, all samples from non infected mice were negative or did not produce coagglutination. This method might be used in the rapid etiological diagnosis also in human cases of acute toxoplasmosis