Navegando por Palavras-chave "estroma"

Agora exibindo 1 - 3 de 3
  • Imagem de Miniatura
    Item
    Acesso aberto (Open Access)
    Assinaturas estromais e expressão de microRNAs relacionados à angiogênese em linfomas não-Hodgkin difuso de grandes células B
    (Universidade Federal de São Paulo (UNIFESP), 2014-11-26) Borges, Natalia Morais [UNIFESP]; Colleoni, Gisele Wally Braga [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Diffuse large B-cell lymphoma (DLBCL) is the most frequent aggressive non-Hodgkin lymphoma (NHL) and, despite advances in treatment, 30% of the cases are refractory or relapse after chemoimmunotherapy. Currently, the relationship between angiogenesis and angiomiRs in DLBCL is unknown. We classified 84 DLBCL according to stromal signatures using tissue microarray and immunohistochemistry (CD34, CD68 and SPARC). We also evaluated the expression of pro- and antiangiomiRs in paraffin embedded tissues of DLBCL by real-time PCR and correlate them with MVD. Approximately 40% of cases were classified as stromal-1, 50% were classified as stromal-2 and 10% were not classified. We observed increased expression of pro-angiomiRs Let-7f, miR-17, miR-18a, miR-19b, miR-126, miR-130a, miR-210, miR-296 and miR-378 in 14%, 57%, 30%, 45%, 12%, 12%, 56%, 58% and 48% of cases, respectively. Among antiangiomiRs we found decreased expression of miR-16, miR-20b, miR-92a, miR-221 and miR-328 in, respectively, 27%, 71%, 2%, 44% and 11%. When comparing the expression of angiomiRs with molecular subtypes according to the algorithm Hans et al. (2004) we found association between increased expression pro-angiomir miR-126, pro-angiomir miR-130a e antiangiomir miR-328 and the subtype ABC (non-GCB). However, only in the case of angiomiRs miR-16, miR-221 and miR-328 we were able to verify expression values compatible with angiogenesis, i.e., higher levels of the three antiangiomiRs in patients with low MVD and stromal-1 signature. The median overall survival has not been reached, with a maximum follow-up of 146.5 months. We observed worse outcome in Ann Arbor stage III/IV, high IPI and CD34 Quartiles III/IV (automated counting). IPI and CD34 confirmed independent impact on survival of the study group. Patients with high-risk IPI showed probability of evolving to death 3.4 times greater than the rest of the group and patients with higher MVD as automated counting (Quartiles III and IV) had chance of evolving to death two times higher than the rest of the group. MicroRNAs showed no prognostic significance in independent serum samples? cohort. Therefore, the stromal-2 signature was found in 50% of cases. We confirmed association between antiangiomirs miR-16, miR-221 and miR-328 and stromal-1 signature. Four angiomiRs emerge as potential therapeutic targets (differentially expressed in ~50% of cases): pro-angiomiRs miR-17, miR-210 and miR-296 and anti-angiomiR miR-20b. Although the four microRNAs seem to be important in DLBCL pathogenesis, they are not predictive of DLBCL onset or relapse.
  • Nenhuma Miniatura disponível
    Item
    Somente Metadadados
    Avaliação da expressão do gene twist1 no microambiente hipóxico da leucemia mieloide aguda
    (Universidade Federal de São Paulo (UNIFESP), 2015-11-25) Malafaia, Emilia Carolina Oliveira Brandao [UNIFESP]; Kerbauy, Daniella Marcia Bahia [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    TWIST1, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in mesodermal development and organogenesis. Overexpressed TWIST1 has been thoroughly related to epithelial-mesenchymal transition (EMT) in solid tumors and has been described as an emerging poor prognostic factor in hematological neoplasms. Many questions remain to be addressed concerning to the role of TWIST1 in acute myeloid leukemia (AML). The understanding of TWIST1 in leukemia cells and its interaction with microenvironment can offer new insights in regards to physiopathology and therapeutic targets for patients with AML. Objectives: to evaluate the role of stroma contact and hypoxia in TWIST1 expression in myeloid cell lines. 2) To evaluate the functional impact of overexpressing TWIST1 on KG1a and PL21 cells. 3) To evaluate TWIST1 expression in primary cells of AML patients. Methods: In order to mimic bone marrow microenvironment, myeloid cells were co-cultured with mesenchymal HS5 cell line and PO2 1% was established with Smart -Trak ? 2 (Sierra Instruments, Inc.) equipment. Quantitative mRNA was determined using TaqMan ? Universal Master Mix (Applied Biosystems, Foster City, CA) and 3-step standard cycling conditions with sequence-specific primer TWIST1 normalized to the expression of ?-actin. KG1a and PL21 cells were transduced with lentivirus vector carrying e-GFP ("enhanced green fluorescence protein") for stable expression of TWIST1. Transduced cells were sorted by FITC fluorochrome and then verified through western blot analysis with TWIST1 antibody. For quantification of apoptosis, cells were labeled with PE-conjugated antibody using annexin V?phycoerythrin and propidium iodide (BD Biosciences, USA). DAPI (4',6- diamidino-2-phenylindole dihydrochloride) was used to stain DNA and determine cell cycle information. Apoptosis and cell cycle were analyzed by FACS -Becton Dickinson Canto II (BD Biosciences). Statistical analysis was assessed with unpaired t test and Fisher?s exact test. Results: Hypoxia induced TWIST1 mRNA expression in OCIAML3, PL21, KG1a and ML1 cell lines (fold-increased 46.3, 29.8, 12.9 and 2.3 respectively). Cells expressing endogenous TWIST1 protein (OCIAML3 and ML1)! 88!showed resistance to apoptosis in a hypoxic microenvironment (normoxia versus hypoxia: OCI/AML3, 22.6 % vs 11.7% and ML1, 29.8% vs. 7.5%) in contrast, cells not expressing endogenous TWIST1 protein (KG1a and PL21) went to apoptosis in the same conditions. Thus, overexpressing TWIST1 in KG1a and PL21 induced apoptosis protection in hypoxia (KG1a unmodified vs. modified: 17.6 ± 6.3 vs. 2.8 ± 6.3, p=0.04; PL21 unmodified vs. modified: 26.9 ± 10.9 vs. 3.2 ± 0.6, p=0.04). We found increased TWIST1 mRNA levels in bone marrow samples of 18 AML patients (3,329 ± 0,64) compared with 6 healthy controls (1,25 ± 0,62) (p= 0.03). Patients in the highest tertile of TWIST1 expression did not show differences in prognostic and complete remission after treatment compared with patients in low and middle tertile. Conclusion: Our data suggest TWIST1 gene expression protects acute myeloid leukemia cells from apoptosis in a hypoxic microenvironment. Moreover, our results showed increased expression of TWIST1 in AML patients. Thus, TWIST1 is a potential gene involved in leukemogenesis and should be further explored to understand disease biology and potential therapeutic targets.
  • Nenhuma Miniatura disponível
    Item
    Somente Metadadados
    Efeitos da angiotensina ii em modelo in vitro de hematopoiese utilizando estroma estabelecido
    (Universidade Federal de São Paulo (UNIFESP), 2013-05-29) Costa, Maira Maftoum [UNIFESP]; Smaili, Soraya Soubhi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    A angiotensina II (AngII) é um hormônio classicamente relacionado a eventos sistêmicos, como o controle do volume de fluídos, balanço eletrolítico e pressão arterial. Alterações em sua produção ou na ativação de seus receptores também foram capazes de induzir anomalias no processo hematopoiético. O peptídeo parece exercer um efeito mitogênico, dependente da ativação do receptor tipo 1 (AT1), preferencialmente em células imaturas do sistema imune, possibilitando uma recuperação mais rápida do sistema sanguíneo de animas imunossuprimidos. Os mecanismo envolvendo estas ações da AngII ainda não foram completamente elucidados, pois aparentemente a via de sinalização ativada é complexa e dependente do fatores do microambiente medular e extramedular. Com isso, neste estudo investigou-se o efeito modulatório da AngII em um modelo de hematopoiese in vitro, permitindo maior controle sobre inúmeros aspectos deste microambiente. Este modelo envolve o cultivo células hematopoiéticas, derivadas de medula óssea de camundongos C57BL/6 sobre a cultura da linhagem estromal murina S17 por 24h com ou sem 100μM de AngII. A imunofenotipagem das células hematopoiéticas mostrou que a AngII modula a porção imatura da co-cultura apenas em meio com baixa concentração de soro fetal bovino (SFB). O tratamento aumenta a porcentagem de células-tronco hematopoiéticas (CTH) e progenitores comuns mielóide (PCM) enquanto reduz os progenitores eritrócito/megacariocíticos (PEM) sem alterar negativamente as características essenciais a estas células, como a quiescencia e indiferenciação. Com o cultivo dos progenitores em meio semi-sólido, foi observado que a AngII, além de interferir na proliferação, estimula a diferenciação dos PGM/CFU-GM e PEM/BFU-E, aumentado a porcentagem de células maduras da linhagem monocítica (Gr-1+Mac-1+) e eritróide (Ter119+) na co-cultura. Os efeitos observados não forma decorrentes da ativação dos receptores AT1 e AT2, dos segundos mensageiro cálcio e EROs, e da ativação das proteínas das vias da PI3K, PKC, MAPK e JNK. Diante destes dados sugerimos que os efeitos da AngII sobre o sistema hematopoiético são complexos e possivelmente envolvem outras vias de sinalização além das clássicas ativadas pelos receptores AT1 e AT2.