expressão gênica de caspases 3 e 8 em timo e baço de ratas recém-desmamadas e imunossuprimidas por glicocorticóide genic expression of caspases 3 and 8 in thymus and spleen of glicocorticoid-immunossupressed weaned rats

Clicks: 366
ID: 177267
2005
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Abstract
Determinou-se a expressão gênica das caspases 3 e 8 mediante transcrição reversa de mRNA total e reação em cadeia da polimerase (RT-PCR) para avaliar a apoptose em timo e baço de ratas imunossuprimidas por glicocorticóides. Utilizou-se dexametasona para indução da apoptose e atrofia linfóide. Quarenta e cinco fêmeas Wistar recém-desmamadas foram separadas em três grupos: as ratas de A (n=18) e B (n=18) foram tratadas com 250 e 500mg de glicocorticóide, via intramuscular, respectivamente, e as do C (n=9) não foram tratadas. Após 24, 48 e 72 horas, seis animais de cada grupo tratado e três do controle foram anestesiados, pesados e sacrificados. O baço e o timo foram coletados e pesados. Fragmentos dos órgãos foram fixados em formol tamponado a 10% e processados segundo técnica para inclusão em parafina. Os blocos foram seccionados em 5µm, e os cortes corados em hematoxilina e eosina. A análise histopatológica aliada ao peso dos órgãos nas diferentes doses e tempos demonstrou que a dexametasona induziu hipotrofia linfóide, que ocorreu com maior intensidade no tempo de 72 horas em animais do grupo B. Fragmentos de timo e de baço foram imediatamente congelados em nitrogênio líquido para extração de mRNA e DNA. Para a padronização da técnica de RT-PCR, utilizaram-se pool de amostras de mRNA dos animais-controle e pool de mRNA de animais tratados em cada tempo de experimento. A técnica de RT-PCR foi sensível o suficiente para a detecção dos mRNAs que codificam as caspases 3 e 8, e ambas participaram do processo de apoptose induzido por dexametasona.
Expression of caspases 3 and 8 in spleen and thymus of immunosuppressed rats was analyzed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Forty-five weaned female Wistar rats were divided into three groups: A (n=18) and B (n=18) rats were injected with 250µg and 500µg per rat, respectively; and C (n=9) rats were non-treated control animals. After 24, 48 and 72 hours, six animals from A and B groups and three controls were anaesthetized with chloroform, weighed and euthanized. Thymus and spleens were collected, weighed and a sample of each organ was fixed by immersion in 10% buffered formaline and embedded in paraffin. Thin sections (5µm) were stained with HE. Thymus and spleen samples were snap frozen in liquid nitrogen and stored at -80ºC for total RNA and DNA extraction. Apoptotic indices were calculated using sections stained with HE. Apoptotic indices and organs weights showed that hypotrophy happened mainly at 72h post treatment with 500µg/rat. RT-PCR was standardized using a pool of control animals and another from treated animals with A and B doses at each time period, respectively. Specific oligonucleotides for caspases 3 and 8 were drawn to obtain fragments of 271bp and 368bp, respectively. The results demonstrated that technique RT-PCR is sensitive enough to detect caspases 3 and 8 mRNAs and that caspase 3 and 8 participate in the apoptotic process induced by dexamethasone in weaned rats.
Reference Key
batista2005arquivoexpresso Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors ;J.J. Batista;A.S. Martins;L. Moro;A.C. Vasconcelos
Journal Environmental research
Year 2005
DOI
10.1590/S0102-09352005000400006
URL
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0102-09352005000400006
Keywords
rt-pcr apoptosis rat thymus caspase 3 spleen dexamethasone

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