Relação entre as metaloproteinases 2 e 9 no processo de fibrose do músculo esquelético de um modelo experimental para distrofia muscular de Duchenne
Arquivos
Data
2017
Autores
Maziero, Carla 
Orientadores
De Oliveira, Flavia
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: A Distrofia Muscular de Duchenne (DMD) é uma doença genética
recessiva ligada ao cromossomo X e caracterizada pela mutação da proteína distrofina.
A DMD afeta a funcionalidade do indivíduo, com comprometimento de força e massa
muscular. A mutação leva a ciclos de degeneração e regeneração de fibras musculares, o
que leva ao aumento da quantidade de tecido conjuntivo no músculo e conseqüente
fibrose. Sabe-se que as metaloproteinases (MMPs) são uma família de enzimas
proteolíticas zinco-dependentes que participam da degradação de macromoléculas da
matriz extracelular, como o colágeno da membrana basal e da matriz intersticial. A
denominação - gelatinase – relacionada tanto a MMP-2 quanto a MMP-9 se deve pelo
fato dessas MMPs degradarem preferencialmente o colágeno desnaturado. Objetivos:
Devido às conseqüências indesejáveis da DMD, o objetivo do estudo é analisar o tecido
conjuntivo do músculo bíceps braquial do animal mdx e a relação do mesmo com a
imunoexpressão das MMP-2 e MMP-9. Até o presente momento os resultados
relacionados a tais investigações já foram coletados e previamente apresentados como
um trabalho de iniciação científica. No entanto, ainda pretende-se ampliar a revisão da
literatura sobre o tópico bem como realizar a discussão dos dados encontrados para a
compilação de um trabalho de conclusão de curso bem como de um artigo científico.
Metodologia: Foram utilizados 12 camundongos (CEUA-UNIFESP 8165240614),
sendo 6 do grupo controle (C), C57BL10, e 6 do grupo DMD, C57BL10-DMD/mdx,
machos, todos com oito semanas de vida. Após a eutanásia dos animais, foi coletado o
músculo bíceps braquial, o qual foi imediatamente fixado em solução de formalina 10%
tamponada com fosfato durante 24h para a análise histopatológica (Hematoxilina-eosina
e Picro-sírus) e imunohistoquímica (MMP-2 e MMP-9). Através das fotomicrografias
das lâminas coradas em hematoxilina-eosina foi ainda determinada a densidade de
fibras musculares (número de fibras/mm2
), a qual foi analisada através do teste t de
Student, considerando o nível significância p≤0,05. Resultados: A análise
histopatológica (Hematoxilina e Eosina) do músculo bíceps braquial dos animais C
revelou fibras musculares com espaçamento eqüidistante, com tamanho homogêneo e
núcleos periféricos. Já os animais do grupo DMD apresentaram as células com núcleos
centralizados e de diferentes tamanhos além de fibras em processo de degeneração em
meio ao intenso infiltrado inflamatório. A análise do tecido conjuntivo, avaliada através
do Picro-Sírius, revelou que os animais controle apresentaram envoltórios musculares (endomísio e perimísio) com características normais, ou seja, sem espessamentos. Já o
grupo DMD apresentou grandes espessamentos entre as fibras musculares e intenso
processo de fibrose. A análise imunohistoquímica tanto para a MMP-2 quanto para a
MMP-9 mostrou intensa imunoexpressão desses marcadores biológicos nos locais com
processo de fibrose. Os resultados da densidade de fibras musculares (número de
fibras/mm2
) revelaram não haver diferença estatisticamente significante quando
comparadas as médias entre os grupos experimentais avaliados. No entanto, observou-se
grande heterogeneidade na avaliação desse parâmetro para o grupo DMD. Conclusão:
O intenso processo de inflamação e fibrose do músculo bíceps braquial dos animais do
grupo DMD analisado no presente estudo mostrou relação com a imunoexpressão da
MMP-2 e MMP-9
Background: Duchenne Muscular Dystrophy (DMD) is a recessive genetic disorder linked to the X chromosome and characterized by changing the protein dystrophin. DMD affects the individual functionality, hard commitment and muscle mass. The mutation leads to degeneration of muscle fibers and regeneration cycles, which leads to increased amount of connective tissue in the muscle and subsequent fibrosis. It is known that metalloproteinases (MMPs) are a family of zinc-dependent proteolytic enzymes that participate in the degradation of macromolecules of the extracellular matrix such as collagen of the basement membrane and interstitial matrix. The name - gelatinase - related both MMP-2 and MMP-9 is due to the fact that these MMPs preferably degrade denatured collagen. Objectives: Due to the undesirable consequences of DMD, the objective of the study is to analyze the connective tissue of the biceps muscle of mdx animals and the relationship of the same with the immunostaining of MMP-2 and MMP-9. To date the results related to such investigations have been collected and previously presented as a scientific initiation work. However, although it is intended to extend the literature review on the topic and conduct a discussion of the data found for compiling a completion of course work and a scientific article. Methodology: Twelve mice (CEUA-UNIFESP 8165240614) were used, 6 of the control group (C), C57BL10, and 6 of the DMD group, C57BL10-DMD / mdx, males, all eight weeks old. After the euthanasia of the animals, the biceps brachii muscle was collected, which was immediately fixed in 10% phosphate buffered formalin solution for 24 h for histopathological analysis (Hematoxylin-eosin and Picro-Syrus) and immunohistochemistry (MMP-2 and MMP -9). The fiber-muscle density (number of fibers / mm2) was also determined by photomicrographs of hematoxylin-eosin stained slides, which was analyzed using Student's t-test, considering the significance level p≤0.05. Results: Histopathological examination (hematoxylin and eosin) of the biceps muscle of C animals showed muscle fibers with equidistant spacing, with homogeneous size and peripheral cores. The animals of the DMD group had cells with centralized nuclei and of different sizes in addition to fibers in the process of degeneration amid intense inflammation. The analysis of connective tissue, assessed by Sirius red, revealed that the control animals showed muscle wraps (endomysium and perimysial) with normal characteristics, without thickening. But the DMD group showed great thickening of the muscle fibers and intense process of fibrosis. Immunohistochemical analysis for both MMP-2 and MMP-9 to show intense immunostaining of these biological markers at the sites of fibrosis process. The results of the muscle fiber density (number of fibers / mm2) showed no statistically significant difference when comparing the mean between available experimental groups. However, there was considerable heterogeneity in the evaluation of this parameter for the DMD group. Conclusion: The intense process of inflammation and fibrosis of the biceps muscle of animals DMD group analyzed in this study showed a relationship with the immunostaining of MMP-2 and MMP-9
Background: Duchenne Muscular Dystrophy (DMD) is a recessive genetic disorder linked to the X chromosome and characterized by changing the protein dystrophin. DMD affects the individual functionality, hard commitment and muscle mass. The mutation leads to degeneration of muscle fibers and regeneration cycles, which leads to increased amount of connective tissue in the muscle and subsequent fibrosis. It is known that metalloproteinases (MMPs) are a family of zinc-dependent proteolytic enzymes that participate in the degradation of macromolecules of the extracellular matrix such as collagen of the basement membrane and interstitial matrix. The name - gelatinase - related both MMP-2 and MMP-9 is due to the fact that these MMPs preferably degrade denatured collagen. Objectives: Due to the undesirable consequences of DMD, the objective of the study is to analyze the connective tissue of the biceps muscle of mdx animals and the relationship of the same with the immunostaining of MMP-2 and MMP-9. To date the results related to such investigations have been collected and previously presented as a scientific initiation work. However, although it is intended to extend the literature review on the topic and conduct a discussion of the data found for compiling a completion of course work and a scientific article. Methodology: Twelve mice (CEUA-UNIFESP 8165240614) were used, 6 of the control group (C), C57BL10, and 6 of the DMD group, C57BL10-DMD / mdx, males, all eight weeks old. After the euthanasia of the animals, the biceps brachii muscle was collected, which was immediately fixed in 10% phosphate buffered formalin solution for 24 h for histopathological analysis (Hematoxylin-eosin and Picro-Syrus) and immunohistochemistry (MMP-2 and MMP -9). The fiber-muscle density (number of fibers / mm2) was also determined by photomicrographs of hematoxylin-eosin stained slides, which was analyzed using Student's t-test, considering the significance level p≤0.05. Results: Histopathological examination (hematoxylin and eosin) of the biceps muscle of C animals showed muscle fibers with equidistant spacing, with homogeneous size and peripheral cores. The animals of the DMD group had cells with centralized nuclei and of different sizes in addition to fibers in the process of degeneration amid intense inflammation. The analysis of connective tissue, assessed by Sirius red, revealed that the control animals showed muscle wraps (endomysium and perimysial) with normal characteristics, without thickening. But the DMD group showed great thickening of the muscle fibers and intense process of fibrosis. Immunohistochemical analysis for both MMP-2 and MMP-9 to show intense immunostaining of these biological markers at the sites of fibrosis process. The results of the muscle fiber density (number of fibers / mm2) showed no statistically significant difference when comparing the mean between available experimental groups. However, there was considerable heterogeneity in the evaluation of this parameter for the DMD group. Conclusion: The intense process of inflammation and fibrosis of the biceps muscle of animals DMD group analyzed in this study showed a relationship with the immunostaining of MMP-2 and MMP-9
Descrição
Citação
MAZIERO, Carla. Relação entre as metaloproteinases 2 e 9 no processo de fibrose do músculo esquelético de um modelo experimental para distrofia muscular de Duchenne. 2017.44 f. Trabalho de conclusão de curso de graduação (Fisioterapia) - Instituto de Saúde e Sociedade, Universidade Federal de São Paulo, Santos, 2017.