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| DC Field | Value | Language |
|---|---|---|
| dc.contributor | Campagnaro, Bianca Prandi | - |
| dc.contributor.author | Ceschim, Sara Lopes | - |
| dc.date.accessioned | 2020-10-05T12:51:24Z | - |
| dc.date.available | 2020-10-05T12:51:24Z | - |
| dc.date.issued | 2015-06-30 | - |
| dc.identifier.uri | https://repositorio.uvv.br//handle/123456789/238 | - |
| dc.description.abstract | A utilização de células-tronco em terapias vem crescendo exponencialmente nos últimos anos devido suas características de auto-renovação. No entanto, estudos relatam que com o envelhecimento estas células podem sofrer alterações nas suas funções devido ao estresse oxidativo, provocado pelo acúmulo de espécies reativas de oxigênio (EROS), e desencadear efeitos genotóxicos severos. Com o intuito de contribuir para identificação do mecanismo do envelhecimento, em células-tronco, e permitir que estas células sejam utilizadas futuramente na prevenção e tratamento de doenças crônicas e degenerativas em idosos através do transplante autólogo, este estudo teve como objetivo avaliar os efeitos do envelhecimento sobre a produção citoplasmática de EROS, por citometria de fluxo, e consequente fragmentação no DNA, pelo ensaio cometa. Para isso, foram utilizadas células-tronco hematopoiéticas da medula óssea de camundongos de 2 e 24 meses de idade da linhagem C57BL/6 divididos em grupo jovem (n = 6) e idoso (n =4). Primeiramente as células-tronco hematopoiéticas foram isoladas pela técnica de citometria de fluxo e os níveis de ânion superóxido (•O2-) foram mensurados pela mediana da intensidade de fluorescência (MFI), após oxidação do dihidroetidio (DHE). Já para avaliação dos danos ao DNA, os cometas formados foram visualizados por microscopia de fluorescência, através da coloração com brometo de etídio, e analisados por um sistema de imagem avaliando os seguintes parâmetros: porcentagem de DNA na cauda (Tail DNA %) e o momento da cauda (Tail Moment). Os dados foram expressos como média±EPM e analisados pelo Teste t de Student. Os resultados obtidos mostraram que os animais do grupo idoso apresentaram maior produção de •O2- quando comparados aos animais do grupo jovem (Jovem: 1561 ± 138 vs. Idoso: 3089 ± 263, MFI). A análise de genotoxicidade revelou que os animais do grupo idoso apresentaram níveis maiores, dos dois parâmetros, quando comparados aos animais do grupo jovem (Jovem: 3.9 ± 0.8% vs. Idoso: 32.2 ± 2.5%, Tail DNA %) e (Jovem: 0.7 ± 0.2 vs. Idoso: 37.5 ± 2.8, Tail moment). Esses resultados sugerem que o envelhecimento aumenta a produção de EROS e consequentemente há formação de danos no DNA de células-tronco, podendo comprometer suas funções. | pt_BR |
| dc.language.iso | pt_BR | pt_BR |
| dc.subject | Envelhecimento | pt_BR |
| dc.subject | Ânion superóxido | pt_BR |
| dc.subject | Estresse oxidativo | pt_BR |
| dc.subject | Danos ao DNA | pt_BR |
| dc.subject | Ensaio cometa | pt_BR |
| dc.subject.vocabulary | CNPQ::CIENCIAS DA SAUDE::FARMACIA | pt_BR |
| dc.subject.vocabulary | CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA | pt_BR |
| dc.title | Análise de genotoxicidade em células - tronco hematopoiéticas da medula óssea de camundongos | pt_BR |
| dc.type | Dissertation | pt_BR |
| dc.publisher.country | brasil | pt_BR |
| dc.description.resumo | In the last decades, stem cells therapy emerged as an alternative to conventional treatment due to its particular characteristics. However, stem cells of intensely regenerative organs and tissues are more susceptible to the cellular damage caused by both intrinsic and extrinsic stressors. Although the response to this process in hematopoietic stem cells (HSC) is crucial, the mechanisms by which hematopoietic homeostasis is sustained are not completely understood. The genes involved in genomic integrity and transcriptional regulation are downregulated in aged cells as a consequence of the accumulation of reactive oxygen species (ROS), which could trigger genotoxicity. In order to determine aging effects on hematopoietic stem cells functionality, in this study, we assessed ROS production and DNA damage, in young and aged (2-, and 24-month-old, respectively) C57BL/6 mice. First, mice were euthanized and the femurs and tibias were removed. The marrow cavities were flushed out and the cell suspension were placed in culture with DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. To enrich the hematopoietic progenitor cell fraction, lineage committed cells were depleted using the Mouse Hematopoietic Stem Cell Enrichment Set (BD) and the Lin- cells were stained with monoclonal antibodies conjugated to different fluorochromes (CD90 (APC), CD133 (PE), SCA1 (FITC) e CD117 (PE-Cy7)). ROS analysis was performed by flow cytometry. Dihydroethidium (DHE, 160 μM) were added to the cell suspension (106 cells) and incubated at 37°C for 30 min in the dark to estimate the intracellular •O2- concentration. The cells were then washed, resuspended in PBS and analyzed by flow cytometry (FACSCanto II). The data were acquired using the FACSDiva software (BD) and overlay histograms were analyzed using FCS Express software trial (De Novo). The cells were excited at 488 nm; DHE fluorescence was detected using a 585/42 bandpass filter. The data are expressed as the median fluorescence intensity (MFI). DNA damage was assessed using the alkaline comet assay. In brief, histological slides were pre-coated with 1.5% normal melting point agarose in PBS in a water-bath at 65°C. Subsequently, 40 μL of the cell suspension was embedded in 100 μL of 0.5% low melting point agarose in PBS at 37°C and spread on agarose-precoated slides using coverslips. Then, the slides were placed in an electrophoresis chamber filled with freshly prepared alkaline buffer (300 mM NaOH, 1 mM EDTA, pH>13) for 20 min at 4°C, and electrophoresed at 300 mA and 20 V for 30 min. Subsequently, the slides were neutralized with a 0.4 M Tris buffer (pH 7.5) for 5 min, washed with cold distilled water and allowed to dry at room temperature overnight. Migration of the DNA fragments towards the anode creates a comet ‘tail’ as visualized by staining with ethidium bromide (20 μg/mL, Sigma-Aldrich). Immediately afterwards, images were obtained at a magnification of 200x using a fluorescence optical microscope (Nikon Eclipse Ti, Melville, NY, USA) equipped with excitation (510-550 nm) and barrier (590 nm) filters. The coded images were acquired using a CCD camera (Nikon) and analyzed using the CASP program (public domain). Data are presented as mean ± SEM. Student’s t test were performed using Graphpad prism 6 software to establish statistical significance between experimental groups at the p <0 .05 (*) level. Aged HSCs showed an increase in intracellular •O2- (3089 ± 263, MFI) compared with young cells (1561 ± 138, MFI). In addition to increased ROS production, we observed augmented DNA damage in aged mice determined by tail DNA % (Young: 3.9 ± 0.8% vs. Aged: 32.2 ± 2.5%) and tail moment (Young: 0.7 ± 0.2 vs. Aged: 37.5 ± 2.8) compared with young mice. These data suggest that this condition affects HSC homeostasis and contributes to increased DNA damage which leads to the premature exhaustion of self-renewal in these cells. In addition, aging impairs the functionality and quality of HSCs and that these age-associated alterations may affect the efficacy of aged HSC recovery and transplantation. | pt_BR |
| Appears in Collections: | Dissertação de Mestrado | |
Files in This Item:
| File | Description | Size | Format | |
|---|---|---|---|---|
| DISSERTAÇÃO FINAL DE SARA LOPES CESCHIM.pdf | DISSERTAÇÃO FINAL DE SARA LOPES CESCHIM | 1.04 MB | Adobe PDF |  View/Open |
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