|Budget Amount *help
¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1998 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1997 : ¥1,400,000 (Direct Cost : ¥1,400,000)
We have explored relationship between mutant mitochondrial DNA (mtDNA) and degeneration of neuronal cells and muscle cells. In this project, firstly, we defined relationsbip between mutant mtDNA and muscle pathology (ragged-red fibers and cytochrome c oxidase-deficient fibers) in MERRF, using single muscle fiber PCR and in situ hybridization. Secondary, we performed an experiment to observe an effect of cytochronme c (Cyt-C) for apoptosis, in which various concentrations of Cyt-C were added in human peripheral blood mononuclear cells and cultured myoblasts from mouse and the cells were observed whether they are induced apoptosis. They did not show apoptosis, resulting from no morphological change and DNA fragmentation, in contrast to the cells induced apoptosis by actynomicin D.Since the morphological change and DNA fragmentation derives from the final step of apoptosis (in three different phases : initiation, effector and degradation), we would like to see if expression of apoptosis r
elated proteins is infulenced at effector phase. For this purpose, we tried to establish methods of immunoblot (IB) and immunohistochemistry (IHC) using antibodies for apoptosis related proteins, and RT-PCR.We purchased various antibodies from several companies, and tested. IB detected bands in control cells for Bcl-2, ICH-1L, TIAR and Bad, but not for Bax and Bcl-X.We also made primer pairs for Bcl-2 and Bax to detect mRNA using RT-PCR.We could detect mRNA for Bcl-2 and Bax using positive control cells. However, we could not detect mRNA for Bcl-2 and Bax in PBMC.We also tried to detect Cyt-C using IHC in muscle sections from patients with mitochondrial encephalomyopathy. However we could not find any Cyt-C positive muscle fibers. This project is now going on establishment of methods for IB.IHC and RT-PCR.After establishment of the methods, we would like to define whether apoptosis induced by Cyt-C can occur in mitochondrial encephalomyopathy.
(2) 特にBcl-2,Baxに対するprimer pairを作成し,RT-PCRの基礎実験を行い,陽性コントロール細胞では各々mRNAを検出できた。PBMCではBcl-2,BaxmRNAを検出できなかった。発現量あるいは感度の問題か,更なる検討を必要とした。