|Budget Amount *help
¥2,700,000 (Direct Cost : ¥2,700,000)
Fiscal Year 1998 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1997 : ¥2,200,000 (Direct Cost : ¥2,200,000)
Present research project was intended to control the acceleration of aging process by introducing genes of various enzymes, which are involved in the reduction of lipid peroxides, to cultured cells and animal body and thereby increasing in lipid peroxide decomposing capability. For this purpose, the investigators of this research project paid a special attention on phospholipid hydroperoxide glutathione peroxidase (PHGPX), since PHGPX is capable of attacking hydroperoxides of membrane phospholipids and located in mitochondria. During the term of this project, investigators successfully found the advance in knowledge as detailed below :
1) When guinea pig driven 104C1 cells, which do not exhibit PHGPx, were incubated with phospholipid hydroperoxides, mitochondrial inner membrane potential (ΔΨm) markedly dissipated. On the contrary, transfectants 104C1/O4C cells, which express high PHGPx, maintained ΔΨm even in the presence of phospholipid hydroperoxides in the medium. This fact indicates
a beneficial effect of the expression of PHGPX gene to prevent lipid peroxide-mediated dysfunction of mitochondria.
2) It is known that he mitochondrial acidic phospholipid cardiolipin strongly binds to cytochrome c. However, the investigators revealed that once cardiolipin was peroxidized, the binding between cardiolipin and cytochrome c was dramatically decreased. This result suggests that peroxidation of the mitochondrial cardiolipin could induce a release of cytochrome c from mitochondria into the cytosol, resulting in cell death.
3) PHGPX is capable of reducing cardiolipin hydroperoxides effectively. In addition, PHGPX activity is stimulated by intact cardiolipin. These observations strongly indicate that PHGPX in mitochondria has an important role in preventing peroxidation of mitochondrial lipid.
4) Smooth muscle cells from rat thoracic aorta were transfected with pRetro-Off vector carrying human PHGPX gene, and transfectants expressing its enzyme were selected. This experiment was carried out to open the strategy to prevent the development of intimal proliferation, especially in graft coronary artery. This is still under investigation.
5) A goal of the research project is to deliver PHGPX gene into desired organs and tissues to increase PHGPX activity in an animal model, senescence accelerated mouse. A liposomal formulation previously developed in our institute was not always ideal, because of less efficiency for in vivo delivery of genes. Therefore, investigators studied on the improvement of liposomes to search suitable liposomes with reporter gene and found liposomes consisting improved cationic lipid is effective to deliver and express the reporter gene in mouse liver, heart, and lung. This subject is still under investigation. Less