O papel modulador do gene AIRE - capes

R.S. Cardoso et al. / Molecular Immunology 43 (2006) 464–472 4673. Results3.1. Monitoring thymocyte maturation in FTOCsThe detection of the TRBV8.1-BD2.1 rearranged DNAsegment was evidence for thymocyte maturation in FTOCs.Using a semiquantitative PCR method, it was possible to detectsignificant amounts (P < 0.05) of amplification productsfor both the -actin internal control and the TRBV8.1-BD2.1segment (Fig. 1). The onset of V(D)J recombination occurredat the 14 day pc fetal thymi after 2 days in FTOC (these 2 daysof incubation time correspond to the 16th day of gestation invivo).Gamma irradiation caused a 1.5-fold significant increase(P < 0.05) of the TRBV8.1-BD2.1 segment in 15 day pc fetalthymus incubated for five days in FTOC (corresponding tothe 20th day pc in vivo); however, organ irradiation did notchange the onset of recombination, which also occurred inirradiated 14 day pc fetal thymus (corresponding to the 16thday of gestation in vivo). This dose of gamma irradiation didnot significantly changed cell viability (t-test, P < 0.05) ofFTOCs as evaluated by determination of frequency of apoptoticand necrotic cells (Table 1).3.2. Hierarquical clusteringThe hierarchical cluster analysis of the results obtainedfor a total of 9216 sequences by comparison between irradiatedand non-irradiated FTOCs showed variability in thehybridization signatures of gene expression presented by differentcultures. The dendrogram shows that this variabilityallowed the distinction of samples according to incubationtime, as well as the distinction between control and irradiatedFTOCs (Fig. 2).It was possible to identify several clusters of repressedand induced genes. However, six of them which includedFig. 1. Semiquantitative detection of V(D)J recombination between TRBV8.1 and BD2.1 during thymus development in control and gamma-irradiated FTOCs.Polymerase chain reactions were performed using TRBV8.1 and BD2.1 specific primers together with beta-actin primers as internal control in order to monitorreactions and normalize data. The 30-cycle PCR allowed amplification of the TRBV8.1-BD2.1 rearranged segment (and beta-actin segment) at log-phase. (A)Southern-blot of PCR products. (B) Quantification of hybridizations using a phosphor-imager. Arbitrary normalized values (on the basis of beta-actin values)expressed as digital light units (DLU) as displayed by the OptiQuant ® software.