Growth hormone (GH), insulin-like growth factor I (IGF-I) & triiodothyronine-T3 are essential for normal somatic growth. T3-mechanism of action involves the regulation of specific genes expression through nuclear T3 receptors (TRs). T3 induces GH and IGF-I gene expression and secretion. The use of recombinant GH and IGF-I for the treatment of growth disorders in children has renewed the interest in the study of the effect of them on thyroid function. Our laboratory has studied the impact of GH and IGF-I in vitro and in vivo in animals, on T3 mechanism of action in several tissues. The results showed that GH & IGF-I induced a reduction of T3-specific responses through a down-regulation of TRs and TR-mRNAs at a transcriptional level. In spite of the accuracy of molecular biology methodology for the diagnosis of most growth disorders, the growth therapeutic response to GH or IGF-I is in some cases below to that which is desired. In consonance to our previous results, those variable therapeutics responses could be due to individual susceptibility to GH or IGF-I that may involve a reduction of TR-expression and consequently a lesser T3-tissue response as in the case of bone. TRs are expressed in mononuclear blood cells although at low levels. Considering the feasibility of blood collection, the assessment of TR mRNA expression in peripheral blood mononuclear cells (PBMC) from children under GH or IGF-I treatments may be a powerful tool to follow up the outcome of these therapies. The aim of this study was to set up a feasible method to quantify TR mRNA level in PBMC to predict possible impairments to GH/IGF-I response due to diminished T3-action induced by these hormones. PBMC were purified from blood collected from 30 healthy children (5-15 y) by ficoll-hypaque and total RNA extraction by the Chomczynski technique. TR mRNA was measured by the reverse transcription reaction followed by the polymerase chain reaction (RT-PCR) technique in parallel to GADPH mRNA quantification to normalize results. Specific primers for both mRNAs amplification were designed to reach similar annealing temperatures and different PCR product sizes for the simultaneous assay of both mRNAs. PCR products were resolved in agarose gels and visualized by ethidium bromide staining. Densitometric analysis was done by a computer software (Scion Image). The results indicated that both mRNAs could be amplified with 3ug total RNA by the same PCR protocol, but to amplify both together, GADPH RT reaction was initiated 30 min after TR RT starting, for 30 min more. To conclude, the development of TR mRNA assay in PBMC may be a useful tool to follow up the response to GH and IGF-I treatments in children. We are presently engaged in its usefulness in children under GH or IGF-I treatment for different growth disorders.
