Demonstration of thyrotropin receptor mRNA in orbital fat and eye muscle tissues from patients with Graves' ophthalmopathy by in situ hybridization

There is a controversy regarding whether there are thyrotropin (TSH) receptors in orbital fat and eye muscle tissues that may play a role in the pathogenesis of Graves' ophthalmopathy. To elucidate whether there are TSH receptors in orbital fat and eye muscle tissues in patients with Graves' ophthalmopathy, we applied the method of in situ hybridization in orbital fat and eye muscle tissues obtained during the operation for patients with Graves' ophthalmopathy, to directly detect TSH receptor mRNA. To identify whether the cells with positive TSH receptor mRNA are fibroblasts, we also did vimentin immunoreactivity study. To further prove the transcript does have a full length of TSH receptor, the samples of total RNA preparations, extracted from orbital fat and eye muscle tissues, were used as a template for reverse transcriptase polymerase chain reaction (RT-PCR) using three primer sets to generate cDNA fragments and cloned for sequencing. The results showed that the expression of TSH receptor mRNA was demonstrated in adipocytes and fibroblasts of orbital fat, and perimysial fibroblasts within eye muscle tissues by in situ hybridization and vimentin immunoreactivity study. Also, by using the RT-PCR, cloning and sequencing, we further proved that the transcript does have a full length of TSH receptor. The present study suggested that there are TSH receptors expressed in orbital fat and eye muscle tissues.