| Budget Amount *help |
¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
|
|---|
| Research Abstract |
objectives : The thymuses in myasthenia gravis (MG) have either thymic hyperplasia (thymitis) or thymoma. We reported the colocalization of nicotinic acetylcholine receptor (nAChR) and the Fas antigen in thymic epithelial cells by immnunohistochemical study. By immunoblott, a part of antigenicity of nAChR was absorbed by Fas. The Homology search showed that the sequence of amino acids of nAChR includes T-cell epitopes, and this portion of homology is also share with the amino acid sequence of the Fas (Fukuoka Acta Med. in press). In this search project, the trigger mechanism of MG was studied by PCR in situ hybridization of intrathymic nAChR in correlation to Fas.
Methods & Results : The presence of nAChR and the Fas in the thymus extract was detected by ECL-Western blot analysis. In May 2000, we are reporting about the Fas and the nAChR in cultured thymus cells. We tried and reported about the isolation of RNA, cDNA synthesis, reverse transcriptase-polymerase chain reaction (RT-PCR) of alpha and gamma subunits of nAChR in rhabdomyosarcoma cell line (TE-671). Based on these results and using GeneAmp In Situ PCR System 1000, we started the study on PCR in situ hybridization of nAChR using biotin labeled deoxynucleotide. The amplified nAChR-DNA was detected by application of labeled streptoavidin-biotin (LSAB) method. The further study has been under way.
Conclusion : By immunohistochemical study nAChR was found in thymic epithelial cells of both cortex and meddulla. By PCR in situ hybridization, the localization was different according to the subunits.
|
