RESUMO
BACKGROUND: Chronic myeloproliferative disorders (MPDs) are clonal haematopoietic stem cell malignancies characterised by an accumulation of mature myeloid cells in bone marrow and peripheral blood. Deregulation of the apoptotic machinery may be associated with MPD physiopathology. AIMS: To evaluate expression of death receptors' family members, mononuclear cell apoptosis resistance, and JAK2 allele burden. SUBJECTS AND METHODS: Bone marrow haematopoietic progenitor CD34 cells were separated using the Ficoll-hypaque protocol followed by the Miltenyi CD34 isolation kit, and peripheral blood leukocytes were separated by the Haes-Steril method. Total RNA was extracted by the Trizol method, the High Capacity Kit was used to synthesise cDNA, and real-time PCR was performed using SybrGreen in ABIPrism 7500 equipment. The results of gene expression quantification are given as 2(-ΔΔCt). The JAK2 V617F mutation was detected by real-time allelic discrimination PCR assay. Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll-hypaque protocol and cultured in the presence of apoptosis inducers. RESULTS: In CD34 cells, there was mRNA overexpression for fas, faim and c-flip in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), as well as fasl in PMF, and dr4 levels were increased in ET. In leukocytes, fas, c-flip and trail levels were increased in PV, and dr5 expression was decreased in ET. There was an association between dr5 and fasl expression and JAK2V617F mutation. PBMCs from patients with PV, ET or PMF showed resistance to apoptosis inducers. CONCLUSIONS: The results indicate deregulation of apoptosis gene expression, which may be associated with MPD pathogenesis leading to accumulation of myeloid cells in MPDs.
Assuntos
Apoptose/genética , Transtornos Mieloproliferativos/metabolismo , Receptores de Morte Celular/biossíntese , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD34/análise , Apoptose/fisiologia , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Humanos , Janus Quinase 2/genética , Leucócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Transtornos Mieloproliferativos/genética , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , Receptores de Morte Celular/genética , Adulto JovemRESUMO
Apoptosis deregulation might have a role in the pathophysiology of polycythemia vera (PV). This study evaluated Bcl-2 molecule expression in CD34+ cells and leukocytes in 12 PV patients. Gene expression was investigated by real time PCR using SybrGreen Quantitect kit and protein expression was evaluated by western-blotting. JAK2 V617F mutation was detected according to Baxter et al (2005). CD34+ cells from PV patients presented higher levels of A1 and Mcl-1 expression (median: 22.6 and 5.2, respectively) in comparison with controls (0.9 and 0.5, p=0.004 and p=0.020); while Bcl-2 and Bcl-xL expression decreased in PV patients (0.18 and 1.19) compared with controls (1.39 and 2.01, p=0.006 and p=0.020). CD34+ cells in PV patients showed an elevated Bid expression (14.4) in comparison with healthy subjects (1.0; p=0.002). Patients' leukocytes showed an A1 augmentation (7.41, p=0.001) and a reduced expression of Bax (0.19; p=0.040) and Bad (0.2; p=0.030). There was no correlation between JAK2 V617F allele burden and molecular expression. PV patients showed alterations in Bcl-2 members' expression, which may interfere with control of apoptotic machinery and contribute to disease pathogenesis.
A desregulação da apoptose parece participar da fisiopatologia da policitemia vera (PV). Este estudo avaliou a expressão das moléculas da família Bcl-2 em células hematopoéticas CD34 + e leucócitos de 12 pacientes com PV. Foram realizados: a quantificação da expressão gênica por PCR em tempo real utilizando kit Sybrgreen Quantitect, avaliação da expressão de proteínas por western-blot e detecção da mutação JAK2 V617F segundo Baxter et al. (2005). Células CD34 + dos pacientes com PV apresentaram maior expressão de A1 e Mcl-1 (mediana: 22,6 e 5,2, respectivamente) em comparação com controles (0,9 e 0,5, p = 0,004 e p = 0,020) e expressão de Bcl-2 e Bcl-xL diminuída nestes pacientes (0,18 e 1,19) em relação aos controles (1,39 e 2,01, p = 0,006 e p = 0,020). Células CD34 + dos pacientes com PV mostraram expressão elevada de bid (14,4) em comparação aos controles (1,0; p = 0,002). Leucócitos dos pacientes mostraram aumento de A1 (7,41, p = 0,001) e expressão reduzida do Bax (0,19; p = 0,04) e Bad (0,2; p = 0,030). Não houve correlação entre percentagem de alelos JAK2 V617F mutados e expressão molecular. Pacientes com PV apresentaram alterações na expressão de moléculas Bcl-2 que podem interferir no controle da apoptose e contribuir para a patogênese da doença.
Assuntos
Humanos , Policitemia Vera/classificação , Apoptose/fisiologia , Genes bcl-2 , MutaçãoRESUMO
OBJECTIVE: High-dose chemotherapy (HDCT) followed by autologous stem cell transplantation is a widely applied treatment for hematological and autoimmune diseases. Little is known about the effects of this therapy on multipotent mesenchymal stromal cells (MSCs). We aimed to characterize, morphologically and functionally, MSCs isolated from bone marrow aspirates of patients after HDCT. MATERIALS AND METHODS: We studied 12 consecutive lymphoma patients submitted to BEAM conditioning regimen followed by autologous stem cell transplantation 28 to 1836 days before the sample collection. Thirteen normal donors were used as control. MSCs were isolated by adherence to plastic and expanded ex vivo by culture in flasks containing alpha-minimum essential medium plus 15% fetal bovine serum. RESULTS: The cell population isolated showed a typical MSC morphology, immunophenotype, and differentiation capacity into adipogenic, osteogenic, and chondrogenic lineages. The MSCs obtained from patients with Hodgkin's disease and non-Hodgkin's lymphoma showed decreased fibroblastoid colony-forming unit count (p = 0.023) and increased doubling time (p = 0.031) related to the control group. The total cell expansion of MSCs from normal subjects was marginally superior to the patient group (p = 0.064). There were no differences in gene expression profile, MSCs plasticity, or hematopoiesis support capability between control and patient group. CONCLUSIONS: Results suggest that HDCT applied to lymphoma patients damaged MSCs, which was demonstrated by their reduced clonogenic potential, doubling time, and cell expansion rates when compared to controls.
Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica , Células da Medula Óssea/efeitos dos fármacos , Doença de Hodgkin , Linfoma não Hodgkin , Células-Tronco Mesenquimais/efeitos dos fármacos , Adulto , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Linfoma não Hodgkin/patologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
OBJETIVOS: O objetivo foi investigar os aspectos etiológicos da esterilidade e/ou infertilidade masculina humana. CASUISTICA E MÉTODOS: Foram avaliados clínica, histopatológica e citogeneticamente 20 indivíduos estéreis que apresentavam hipótese diagnóstica de alteraçäo testicular. O estudo citogenético foi realizado em células somáticas (cultura de linfócitos) e em células germinativas (foram estudados os cromossomos meióticos a partir da biópsia testicular). RESULTADOS: As patologias genéticas foram detectadas em 35 por cento e as näo genéticas (ambientais) em 15 por cento dos pacientes, sendo que em 50 por cento da amostra näo foi possível a elucidaçäo da causa da alteraçäo da fertilidade através da metodologia utilizada. Entre os 7 casos de patologias genéticas, três corresponderam a doença monogênica que altera o pareamento cromossômico meiótico (dissinapse), três pacientes apresentaram a doença monogênica da aplasia de células germinativas e uma paciente apresentou uma aberraçäo cromossômica clássica (mosaico 46,XY/47,XY,+mar). Na etiologia ambiental ficou evidenciada a importância dos fatores infecciosos. CONCLUSöES: Este estudo mostra a importância do estudo clínico, histopatológico e genético na elucidaçäo da etiologia da esterilidade e/ou infertilidade masculina, antes do paciente ser submetido as técnicas de reproduçäo assistida.