EXPRESSION OF THYROID HORMONE RECEPTOR mRNA DURING THE HUMAN BRAIN DEVELOPMENT / 解剖学报

Objective To observe the variations of thyroid hormone receptors(RTs) mRNA experession during the human brain devlopment. Methods We investigated the ontogeny of TR isoforms in the first and second trimester human fetal different brain areas by semi-quantitative reverse transcriptase-polymerase chain reaction analysis. When we amplified the TR? 2 by PCR, the other sequence was amplified at the same time, it is about 100pb less than the RT? 2, so we cloned it into pGEM-T easy vector to determine its sequence. Results In the first and second trimester human fatal brain, RTs mRNAs were detected in cerebrum, cerebellum, brain, stem, hippocampus, spinal cord, thalamus. TRs mRNAs were relatively higher in cerebrum, cerebellum, hippocampus. In the first trimester human fatal brain, the TR? isoforms mRNAs were higher than TR? 1, In the second trimester human fatal brain, the TR? 2 and TR? 1 were higher than TR? 1. An additional truncated species was detected with the TR? 2 primer set. We submitted its sequencing results to Genbank, comparing it with TR? 2 by BLAST software, the results showed that it is identical to TR? 2 with the exception that it is missing 42 amino acids at 371-412 of TR? 2 sequence, so it is the human TR? 3. At the same time we acquired the Genbank accession number AF522368. Conclusion The spatial and temporal expressions of TR isoforms mRNAs exist in CNS development. We firstly assure the different sequence between human TR?2 and TR?3.

Human genomic DNA isolation and chromosomal localization of fetal brain cDNA (FB174) / 대한해부학회지

Eventhough surmountable amounts of genes are being cloned and a number of methods are being developed by human genome project, it's not easy to predict possible functions of genes and determine the chromosomal locations of genes. In this experiment, cDNA pool was made from 18 weeks old human fetal brain and analyzed the sequences. FB174 clone was chosen, in situ hybridization histochemistry was performed on developing and adult rat tissue section to observe the tissue specificity and developmental expression of this gene. To observe the chromosomal location of FB174 clone, the genomic DNA from human genomic library was isolated and fluorescence in situ hybridization was carried out. By sequencing and sequence search with GenBank data it was revealed that cloned FB174 cDNA was quite similar to translationally controlled tumor protein which is known to locate to human chromosome 13q14. The expression of FB174 mRNA was not detected in rat tissue sections by in situ hybridization histochemistry. Fluorescence in situ hybridization using biotin labeled FB174 probe resulted in specific labeling of human chromosome 7q22. These results and high sequence homology of FB174 to known translationally controlled tumor protein suggest that FB174 clone may be a new translationally controlled tumor protein-related gene.

A Study on the Formation of Organotypic Spheroids from Early Human Fetal Brain

The present study investigated whether organotypic spheroids derived from human fetal brain tissue, cultured at agarose-overlay media, may serve as an optimal invitro model for multidisciplinary studies in human neurobiology, particularly in the fields of tumor invasiveness and its biochemical mechanism, using light microscopy, electron microscopy and immunohistochemical staining. Eight fetal brain tissues of 8-9 weeks of gestation were minced and explanted into agarose-coated culture wells. After three to five days these human fetal brain tissue fragments emerged as spheroids and could be maintained as organotypic spheroids for up to seven weeks. Light and electron microscopic studies of sphereoids demonstrated that most cells were poorly differentiated and there were no definite mature neurons or glial cells after enough culture time, but some cells showed certain evidence suggestive of differentiation to neurons or glial cells. Immunohistochemical staining for glial fibrillary acidic protein(GFAP) and neur on specific enolase(NSE) demonstrated that NSE-positive cells were oval or spherical cells containing abundant cytoplasm and GFAP-positive cells were fibrillary cytoplasma-containing cells which showed some evidence suggestive of differentiation to glial cells by light microscopy. In future, adding some modifications in culture, this organotypic spheroids derived from the human fetal brain may serve as an optimal in vitro model for neurobiology especially in the field of studies on tumor invasiveness through co-culture with microtumor spheroids.