RESUMO
RESUMEN: Las enfermedades cardiovasculares (ECV) son la principal causa de muerte a nivel mundial, donde la terapia con Células Troncales Mesenquimales (CTM) representa una alternativa para los pacientes que no logran recuperarse con los tratamientos actuales. El lograr que las CTM residentes se movilicen al órgano afectado representaría una ventaja para el manejo terapéutico de las ECV. La dehidroepiandrosterona (DHEA) es un precursor hormonal cuyos niveles disminuyen a lo largo de la vida, lo que se ha asociado al desarrollo de ECV. Diversos estudios han demostrado que el consumo de DHEA previene y mejora la condición cardiaca, aunque no se sabe si esto ocurre porque se ejerce un efecto en los cardiomiocitos y estos, a su vez, hacia las CTM. El objetivo del presente estudio fue determinar el efecto del medio condicionado procedente de la línea H9C2 pretratada con DHEA y sometida a daño, sobre la motilidad de CTM, llevando a cabo un ensayo de cierre de herida. El pretratamiento con DHEA y el daño en la línea H9C2, promueve la motilidad de CTM. El estímulo de la motilidad de CTM por un efecto indirecto de DHEA podría ser una estrategia terapéutica para el daño cardiaco.
ABSTRACT: Cardiovascular diseases (CVD) are the leading cause of death worldwide. Mesenchymal Stem Cell (MSC) therapy is an alternative for patients who cannot recover with current treatments. Ensure movilization of MSC to the affected organs would represent an advantage for therapeutic management of CVD. Dehydroepiandrosterone (DHEA) is a hormone precursor whose levels decrease throughout life, which has been associated with the onset of CVD. Several studies have shown that DHEA consumption, prevents and improves heart condition, although it is not known if this is because an effect on cardiomyocytes is exercised on these cells and this, in turn, to CTM. The aim of this study was to determine the effect of conditioned medium from H9C2 cell line pretreated with DHEA and subjected to damage, on the motility of CTM, performing a wound healing assay. Pretreatment with DHEA and damage to H9C2 cell line, promotes motility of CTM. Stimulation of CTM motility by an indirect effect of DHEA could be a therapeutic strategy for heart damage.
RESUMO
RESUMEN: Las células troncales mesenquimales (CTM) representan una población heterogénea con capacidad para auto-renovarse y diferenciarse a distintos tipos celulares. Estas fueron descritas en un inicio en médula ósea (MO) a mediados del siglo pasado, desde entonces este tejido se ha convertido en el estándar de oro para la obtención y caracterización de CTM. Actualmente se sabe que este tipo de células se encuentran alojadas en nichos distribuidos por todo el organismo, donde contribuyen a los procesos de regeneración del tejido donde se localizan. No obstante, encontrar una fuente alterna de CTM con las mismas características que las de MO, pero que su extracción no suponga riesgo para el donador es fundamental para su utilización con fines terapéuticos. En este trabajo se aislaron células troncales de médula ósea, y se compararon con tejido adiposo y gelatina de Wharton y caracterizaron de acuerdo a los criterios de la Sociedad Internacional para la Terapia Celular (ISCT). Los resultados mostraron que la morfología, diferenciación osteogénica y adipogénica, así como la expresión de los antígenos de superficie CD90, CD73 y CD105 cumplen con los estándares, señalando a las provenientes de gelatina de Wharton como mejor opción.
ABSTRACT: Mesenchymal stem cells (MSC) represent a heterogeneous population with the capacity to self-renew and differentiate into different cell types. At the middle of the last century these cells initially were described in bone marrow (BM), thence this tissue has become the gold standard for obtaining and characterization of MSC. It is known that these cells are housed in specific areas called niches distributed throughout all body, where they contribute to tissue regeneration processes of self-tissue were they are located. However, finding an alternative source of CTM with the same characteristics that have showed in MO, but its obtention no represent a risk since the donor is essential to their use for therapeutic purposes. In this study we isolated mesenchymal stem cells from bone marrow, adipose tissue and Wharton's jelly and they were compared in their characteristics in according to the standards of the International Society for Cellular Therapy (ISCT). The results showed that the morphology as well as adipogenic and osteogenic differentiation and also the expression of surface antigens (CD90, CD73, and CD105) from all tissues accomplished the standards, although Wharton's jelly represented the best option.
RESUMO
RESUMEN: La piel es el órgano más extenso en el ser humano, su integridad representa protección contra diferentes agentes químicos, biológicos y mecánicos. Las lesiones ocasionadas en este tejido se resuelven mediante la formación de una cicatriz, sin embargo, diferentes alteraciones moleculares pueden sobre estimular este proceso, lo que conlleva a la formación de cicatrices aberrantes (hipertrófica o queloide). El tratamiento más recomendado para este tipo de lesiones es la aplicación intralesional del acetónido de triamcinolona (AT) y por otro lado, la dehidroepiandrosterona (DHEA) es una pro-hormona que posee una gran variedad de efectos biológicos como: regulación de la síntesis de fibras de colágeno, protección celular, propiedades antitumorales, antiinflamatorias y antioxidante. En este trabajo, se estudió la combinación de AT-DHEA sobre la proliferación y muerte celular en la línea celular de fibroblastos 3T3-L1. Los resultados mostraron que la AT a 100 M y la DHEA a 1000 M inhiben la proliferación en un 50 y 40% respectivamente. La combinación de AT-DHEA (10000-10 M) inhibe la proliferación celular e inducen muerte celular programada, entonces esta combinación pudiera utilizarse en cicatrices hipertróficas o queloides para su eliminación.
ABSTRACT: The skin in the human is the largest organ, his integrity represents protection against various chemical, biological and mechanical agents. The injuries in this tissue are solved by forming a scar, however, different molecular alterations may overstimulate this process, leading to the formation of aberrant scars (hypertrophic or keloid). The most recommended treatment for such injuries is the intralesional application of triamcinolone acetonide (TA) and on the other hand, dehydroepiandrosterone (DHEA) is a pro-hormone that has a wide variety of biological effects such as regulation of the synthesis of collagen fibers, cell protection, anti-tumor properties, anti-inflammatory and antioxidant. In this paper, the combination of AT-DHEA on proliferation and cell death in fibroblast cell line 3T3-L1 was studied. The results showed that the AT 100 and 1000 M DHEA to inhibit proliferation by 50 and 40% respectively. The combination of AT-DHEA (10000-10 M) inhibits cell proliferation and induce programmed cell death, so this combination could be used in hypertrophic or keloid scars for disposal.
RESUMO
Identification of organic compounds from plants is of clinical significance because of the effect that they might have in patients with haematopoietic disorders. We studied the effect of the plant extract Justicia spicigera (Acanthaceae) in different haematopoietic cells: human leukaemic cell lines, umbilical cord blood cells, and mouse bone marrow cells. By examining colony formation and performing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay it was shown that the plant extract of Justicia spicigera contains cytotoxic factors for leukaemic cells and has no proliferative activity on normal haematopoietic progenitor cells. Our results show that this plant extract induces apoptosis in the human leukaemia cell line TF-1, but not in the bcl-2 transfectant cell line TB-1. Similar results were obtained using a haemopoietic cell line 32D and 32DBcl2. The cultures of umbilical cord blood cells and mouse bone marrow that contain granulocyte-macrophage colony-stimulating factor (GM-CSF) do not proliferate or become terminally differentiated in the presence of the infusion of Justicia spicigera. GM-CSF that acts by abrogating programmed cell death is not sufficient to inhibit the apoptotic stimulus in TF-1 and 32D cells. Moreover mouse fibroblasts (3T3) and two cervical carcinoma cell lines CALO and INBL, undergo apoptosis in the presence of different concentrations of an infusion from the plant. Our data show that there is a strong correlation between the cytotoxic effect and cell proliferation. Together, these results indicate that the plant infusion of Justicia spicigera does not contain any haematopoietic activity, induces apoptosis inhibited by bcl-2 and is linked to cell proliferation.
Assuntos
Acanthaceae , Ciclo Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/toxicidade , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Feminino , Sangue Fetal/citologia , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Estruturas Vegetais , Proto-Oncogene Mas , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Cell-cell interaction is important in the expansion of leukemic cells and of solid tumors. Steel factor (SF) or Kit ligand is produced as a membrane-bound form (mSF) and a soluble form. Because both primary gynecological tumors and primary leukemic cells from patients with acute myeloblastic leukemia (AML) have been shown to coexpress c-Kit and SF, we addressed the question of whether mSF could contribute to cell interaction in these cancers. Investigations on primary cervical carcinomas have been hindered by the fact that the cells do not grow in culture. We report herein the establishment of two cervical carcinoma cell lines, CALO and INBL, that reproduce the pattern of SF/c-Kit expression observed in primary tumor samples. In addition, these cells exhibit marked density-dependent growth much in the same way as AML blasts. Using an antisense strategy with phosphorothioate-modified oligonucleotides that specifically target SF without affecting other surface markers, we provide direct evidence for a role of mSF and c-Kit in cell interaction and cell survival in these gynecological tumor cell lines as well as in primary AML blasts. Finally, our study defines the importance of juxtacrine stimulation, which may be as important, if not more, than autocrine stimulation in cancers.
Assuntos
Comunicação Celular/fisiologia , Leucemia Mieloide/patologia , Proteínas Proto-Oncogênicas c-kit/fisiologia , Fator de Células-Tronco/fisiologia , Neoplasias do Colo do Útero/patologia , Células 3T3 , Doença Aguda , Animais , Contagem de Células , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Chlorocebus aethiops , Feminino , Células HeLa , Humanos , Leucemia Monocítica Aguda/genética , Leucemia Monocítica Aguda/metabolismo , Leucemia Monocítica Aguda/patologia , Leucemia Mieloide/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Leucemia Mielomonocítica Aguda/genética , Leucemia Mielomonocítica Aguda/metabolismo , Leucemia Mielomonocítica Aguda/patologia , Camundongos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas c-kit/biossíntese , Proteínas Proto-Oncogênicas c-kit/genética , Fator de Células-Tronco/antagonistas & inibidores , Fator de Células-Tronco/biossíntese , Fator de Células-Tronco/genética , Tionucleotídeos/genética , Tionucleotídeos/farmacologia , Células Tumorais CultivadasRESUMO
UNLABELLED: Apoptosis is a process genetically controlled. The produce of the bcl-2 gene, bcl-2, is an anti apoptotic protein that is linked to the external membrane of the mitochondria. OBJECTIVE: To explore the possibility that bcl-2 transfection could change phenotype, response to mitogenic factor, and cell morphology on the TF-1 parental cell line and the bcl-2 transfectant TB-1 or TF-1neo. METHODS: We look at the expression of CD13, CD34 and c-Kit surface markers by flow cytometry. We have measured cell proliferation in response to GM-CSF and cell survival after GM-CSF withdrawal by the MTT assay on the same cell lines. Apoptosis was evaluated by the apoptotic membrane blebbing set up at different times after serum and survival factor removal or tolerance to cytotoxic compounds from Justicia spicigera. RESULTS: According with our results, ectopic expression of the bcl-2 gene prevented apoptosis without changes in morphology or phenotype in the absence of GM-CSF and serum or the presence of the extract from Justicia spicigera. Consisting with the Bcl-2 function, we found that Bcl-2 did not change response to GM-CSF. Serum deprivation or GM-CSF withdrawal induces cell death at 36 hours in TF-1 and TF-1neo cells, whereas TB-1 cells undergo apoptotic membrane blebbing after 96 hours under the same conditions. CONCLUSIONS: Taken together, our data indicate that Bcl-2 is a short term anti apoptotic protein in TB-1 cell line, that does not affect response to GM-CSF neither CD13, CD34 nor c-Kit antigen expression.
Assuntos
Genes bcl-2/genética , Transfecção , Linhagem Celular , Sobrevivência Celular , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Humanos , Fenótipo , Proto-Oncogene Mas , Fatores de TempoRESUMO
The regulation of cell differentiation and cell death in crucial to the generation of hematopoietic cells both in vitro and in vivo. The biologic role of stem cell factor (SCF) in hematopoietic cell development is not well known. We monitored the survival, proliferation and differentiation of mouse hematopoietic cells in culture in the presence of SCF. Examination of colony formation, MTT and thymidine killing of mouse bone marrow indicated that SCF is mainly a survival factor. Our results show that SCF maintains cells in a "undifferentiated" state. Committed granulocytic and monocytic progenitors (CFU-GM) survive for seven days in the presence of SCF alone, under conditions where no maturing granulocytic monocytic cells could be recovered. On transfer to GM-CSF containing cultures, these cells proliferate and differentiate terminally. Together, our data indicate that SCF induces survival in hematopoietic progenitors. Furthermore, SCF favors the survival of granulocytic progenitors over that of monocytic progenitors. In the absence of later acting factors such as GM-CSF, cells that progress beyond the CFU-GM stage lose c-kit expression and die by default. Hence, lack of cell expansion in the presence of SCF by itself is due to constant cell proliferation and survival, which is counterbalanced by cell death. In contrast, the presence of both SCF and GM-CSF allows for the continuous survival and expansion of hematopoietic progenitor cells in culture, as well as favoring their terminal differentiation along granulocytic and monocytic pathways. Furthermore, GM-CSF induces colonies of macrophages that produce G-CSF and IL-6, two molecules involved in granulopoiesis, and these in turn stimulate granulocyte colony formation. Finally, our data suggest that survival signals by SCF are crucial during the differentiative process of granulocytes, giving strength to deterministic model.
Assuntos
Apoptose/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Granulócitos/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fator de Células-Tronco/farmacologia , Animais , Divisão Celular , Linhagem da Célula , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Feminino , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Monócitos/citologia , Timidina/farmacologiaRESUMO
All the cells comprising the hemopoietic system are derived from a common precursor, the totipotent hemopoietic cell (THC), which, through processes of proliferation and differentiation, gives rise to all the mature cells found in the blood and lympho-hemopoietic organs. In order that the processes of proliferation, survival, apoptosis, and differentiation from THCs to mature cells take place, the participation of proteins denoted collectively as cytokines is required. Their role is to promote and regulate one or several functions (depending on the cell type and stage of development), and to participate in one or several stages of cell development of the THCs. By the use of different tissue culture techniques, it was concluded that other non-hemopoietic cell types have an important role. These cells are those comprising the stroma in the bone marrow: fibroblasts, endothelial cells and adipocytes among others. The contribution of the stroma lies in the production of cytokines, as well as providing sustenance for the THCs. Even when it could seem that cytokines are fundamental factors in the regulation of the main functions of the hemopoietic cells, two models have been proposed to explain the process of hemopoiesis: the deterministic and the stocastic. Both models provide some evidence to support their postulates, however, to this date it is not possible, in view of the data, to decide which of the models is more accurate without incurring controversy. Even though the study of the THCs promotes a great number of questions about the basic mechanisms that regulate them, in several laboratories in the world a new aspect of research: their use in transplants, using THCs as a substitute for whole bone marrow transplant and, still in the initial stages, their use as targets for gene therapy for deficiency diseases or even for therapy against cancer.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Animais , HumanosRESUMO
Mice bearing mutations at either of two loci, dominant White spotting(W) or Steel(Sl), exhibit development defects in hematopoietic, melanocytic and germ cells. Genetics studies have shown that the SI locus encodes the Steel factor (SF), which is the ligand for the tyrosine kinase receptor c-kit, the product of the W locus. SF is synthesized in membrane-bound form and can be processed to produce a soluble form. Cell-cell interaction is important in the production of normal blood cells in vivo and in vitro and in the cellular expansion of leukemic cells. We discuss here how SF decreases the requirements in cell interaction for blast colony formation in acute myeloblastic leukemia (AML) and the presence of membrane-bound SF possibly contributes to the density-dependent growth of the AML blasts. We explain that SF is mainly a survival factor for hematopoietic cells, of little proliferative effect, which maintains CD34+ hematopoietic cells in an undifferentiated state. These properties would potentially allow the maintenance of hematopoietic cells in culture for the purpose of marrow purging or gene therapy. The activation of the c-kit signal transduction pathway may play a significant role in the development of many types of non-hematological malignancies by disrupting normal cell-cell interactions and allowing the growth of cancer cell populations. In summary, the properties of the SF indicate it has a role for survival signals during the process of normal differentiation, AML proliferation and in the maintenance of many c-kit+ tumors.