| 研究実績の概要 |
We mostly focused on to establish protocol for isolating the exosome from the fish serum and from the mammalian cultured cell medium. The serum were isolated from the blood of adult tilapia by centrifuging the blood samples. The exosomes were identified from the serum of adult tilapia by using both ultracentrifugation and commercially available kits-based protocol. Exosomes were also isolated from the cultured human cancer cells named Hella following standard untracentrifugation and kit-based protocol. After isolating the exosomes, we confirmed their presence by electron microscopy. The size of exosomes was confirmed less than 100 nm by electron microscopy. The western blot was carried out by using exosomal marker-based primary antibodies such as CD63, TSG101 and CD19. The western blot confirmed the positive signal of the isolated exosome from the tilapia serum and Hella cell line by using the above antibodies. Nano-particle tracking system to further confirm the exosome size and concentration from the fish serum and cultured cells are progressing. After standardizing the exosome isolation protocol, our research will focus on the establishment of relationship between the role of exosome and aging in both fish and mammalian models.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
The proposed research plan consists of two main parts that includes (1) the elucidation of the roles of exosomes in aging in fish and mammals, and (2) To use gene expression profiles of skeletal muscles to discover the main genetic pathway associated with indeterminate growth. At present, our research focusing on the elucidation of the roles of exosomes in aging. For this purposes, we are isolating the exosomes from the serum samples of different aged fish and cultured mammalian cells. The exosomes are isolated from the serum of young, adult an aged tilapia and common carps and from young and aging cell culture of human diploid fibroblasts following standard protocol. After exosome isolation, exosomal microRNA will be quantified. Exosomal RNA will be sequenced by using standard protocol. Gene expression analysis will be conducted between young, adult and aged fish serum and, proliferative and aging diploid fibroblast cells exosomes by Real-time PCR. The current research would identify the new and novel roles of exosome-associated RNA in aging.
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| 今後の研究の推進方策 |
Besides the exosome-based study, future research will mostly focus on the gene expression profiles of the skeletal muscle to discover the genetic pathway associated indeterminate growth. The lifelong growth of many teleosts begs this question: does the existence of an asymptotic growth plateau lead to senescence? Studies utilizing the model teleost have provided some insight into this question. In the proposed plan, we will use young zebrafish, aged zebrafish and accelerated aging fish observed in growth hormone transgenic fish for transcriptomic analysis. While zebrafish appear to age much slower, signs of senescence are nonetheless present. However, its kin, the giant danio, displays an apparent resistance to aging (Froehlich et al., 2013). Therefore, the transcriptomic profiling of aged zebrafish and aged giant danio will also be conducted. The proposed research plan will use muscle RNA-seq transcriptomic analysis, micro array analysis, real-time PCR analysis to discover the molecular mechanism of indeterminate growth. Genome-wide changes in gene expression will serve as a starting point to understand the molecular mechanism by which these teleost fish avoid aging related muscular disorder. Therefore, changes that accompany aging of skeletal muscles at will be known. The proposed research will strengthen our understanding about the age-related muscular disorder in mammals.
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