2000 Fiscal Year Annual Research Report

耐凍性タンパク質遺伝子等のex vivo導入による卵巣の凍結保存

Research Project

Project/Area Number	12876061
Research Institution	Kyoto University
Principal Investigator	宮本元京都大学, 農学研究科, 教授 (00026618)
Co-Investigator(Kenkyū-buntansha)	眞鍋昇京都大学, 農学研究科, 助教授 (80243070)
Keywords	凍結保存 / 卵巣 / 耐凍タンパク質 / アポトーシス受容体 / Ex vivo遺伝子導入 / リポソーム法 / アポトーシス阻止因子 / NIH-3T3細胞
Research Abstract	哺乳類の生殖器官の凍結保存を実現するために、薄切した卵巣を急速に冷却する手法が開発されたが、受精能をもつ卵子をほとんど得られなかった。動脈から耐凍剤を灌流した後薄切して凍結する方法が考案されたが、これも満足のいく成績を得られなかった。遊離細胞より格段に巨大な卵巣の凍結保存を実現するためにはブレークスルー的技術革新が必要である。本研究は、極地に生息する動物の体液氷点を低下させたり氷晶表面に結合して結晶の成長を抑えて細胞破砕を阻止する耐凍タンパク質の遺伝子と申請者らが見いだしたアポトーシス阻止遺伝子を卵巣にex vivo導入し、凍害保護や細胞死阻害によって生殖器官の凍結保存を実現しようとするものである。Ex vivo遺伝子導入のためのリポソーム作製法の基礎研究を進めた。卵巣に耐凍性タンパク質と申請者らが発見した新規アポトーシス阻害因子の遺伝子を特異的に発現させるex vivo遺伝子導入法をマウスにて確立している。遺伝子導入に際して遺伝情報の伝達を担う卵子に外来遺伝子が不用意に導入されることを未然に防がなくてはならない。導入効率は高いがウイルス遺伝子を含むウイルスベクター法はとらず、ウイルス遺伝子を全く含まないリポソーム法を開発している。極地の魚類や節足動物の耐凍タンパク質には化学構造の違うI、II、III型とN型糖鎖をもつantifreeze glycoproteinがあり、すでにI型とIII型はクローニングされている。これらのセンスmRNAを調製して培養NIH-3T3細胞にin vitro導入してタンパク質産生能をスクリーニングし、最も産生能の高いクローンを選抜している。併せてI型とIII型をコードずるcDNAを合成して大腸菌プラスミドに組み込んで大量に調製した。同様に、申請者らの発見した新規アポトーシス阻害因子や既知アポトーシス阻害因子p53をコードするmRNAを生殖器官に導入するための導入用遺伝子クローンを組み込んだプラスミドのin vitro選抜を行ている。

Research Products

(14 results)

All Other

All Publications (14 results)

[Publications] Noboru Manabe,Miki Sugimoto,Maki Morita,Hajime Miyamoto et al.: "A novel in vivo 31P-nuclear magnetic resonance technique for assessment of teratogenicity induced by environmental endocrine disrupters in mice."Japanese Journal of Clinical Medicine. 58・2. 108-111 (2000)
[Publications] Noboru Manabe,Miki Sugimoto,Maki Morita,Hajime Miyamoto et al.: "In vivo noninvasive nuclear magnetic resonance techinque for assessment of teratogenicity induced by environmental pollutants."Environment Measurement and Chemical Analysis. 27・1. 60-66 (2000)
[Publications] Noboru Manabe,Yasuyoshi Azuma,Hajime Miyamoto et al.: "Effects of the mycelial extract of cultured Cordy ceps sinensis on in vivo hepatic energy metabolism and blood flow in dietary hypoferric anemic mice."British Journal of Nutrition. 83・2. 197-204 (2000)
[Publications] Noboru Manabe,Akira Myoumoto,Chiemi Tajima,Hajime Miyamoto et al.: "Immunochemical characteristics of a novel cell death receptor and a decoy receptor on granulosa cells of porcine ovarian follicles."Cytotechnology. 33・2. 189-201 (2000)
[Publications] Mizuho Nakayama,Noboru Manabe,Hajime Miyamoto et al.: "Species specific differences in apoptotic cell localization in granulosa and theca interna cells during follicular atresia in porcine and bovine ovaries."Journal of Reproduction and Development. 46・2. 147-156 (2000)
[Publications] Susumu Nishihara,Noboru Manabe,Hajime Miyamoto et al.: "Changes in cell adhesion molecules during follicular atresia in porcine ovaries."Journal of Reproduction and Development. 46・3. 325-334 (2000)
[Publications] Mizuho Nakayama,Noboru Manabe,Hajime Miyamoto et al.: "Changes in localization of type I, III and IV collagens and their mRNA expression during follicular atresia in bovine and porcine ovaries."Journal of Reproduction and Development. 47・1. 27-36 (2001)
[Publications] Kozue Uchio,Noboru Manabe,Kotaro Tamura,Makoto Miyamoto et al.: "Decreased matrix metalloproteinase activity in the kidneys of hereditary nephrotic mice (ICGN strain)."Nephron. 86・1. 145-141 (2000)
[Publications] Kotaro Tamura,Noboru Manabe,Kozue Uchio,Makoto Miyamoto et al.: "Characteristic changes in carbohydrate profile in the kidneys of hereditary nephrotic mice (ICGN strain)."The Journal of Veterinary Medical Science. 52・2. 379-390 (2000)
[Publications] Noboru Manabe,Akiko Kinoshita,Kozue Yamada-Uchio et al.: "Changes in quantitative profile of extracellular matrix components in the kidneys of rats with adriamycin-induced nephropathy."The Journal of Veterinary Medical Science. 53・1. 125-133 (2001)
[Publications] Miki Sugimoto,Noboru Manabe,Hajime Miyamoto.: "Future functional-morphological studies in embryology."Journal of Reproduction and Development. 46・1. 71-77 (2000)
[Publications] Noboru Manabe,Hajime Miyamoto et al.: "Immunohistochemical quantification of fast-myosin in upper hind limb muscles of the pig."Animal Science Journal. 72・2. 154-163 (2000)
[Publications] Miki Sugimoto,Noboru Manabe,Hajime Miyamoto.: "Development of infantile rat ovaries autotransplanted after cryopreservation by vitrification."Theriogenology. 53・11. 1093-1103 (2000)
[Publications] Noboru Manabe,Akira Myoumoto,Chiemi Tajima,Hajime Miyamoto et al.: "A novel cell death receptor and a decoy receptor on granulosa cells of porcine ovarian follicles."Tohoku University Press, Sendai, Japan.. 357 (2000)

2000 Fiscal Year Annual Research Report

耐凍性タンパク質遺伝子等のex vivo導入による卵巣の凍結保存

Principal Investigator

宮本 元 京都大学, 農学研究科, 教授 (00026618)

Research Products

[Publications] Noboru Manabe,Miki Sugimoto,Maki Morita,Hajime Miyamoto et al.: "A novel in vivo 31P-nuclear magnetic resonance technique for assessment of teratogenicity induced by environmental endocrine disrupters in mice."Japanese Journal of Clinical Medicine. 58・2. 108-111 (2000)

[Publications] Noboru Manabe,Miki Sugimoto,Maki Morita,Hajime Miyamoto et al.: "In vivo noninvasive nuclear magnetic resonance techinque for assessment of teratogenicity induced by environmental pollutants."Environment Measurement and Chemical Analysis. 27・1. 60-66 (2000)

[Publications] Noboru Manabe,Yasuyoshi Azuma,Hajime Miyamoto et al.: "Effects of the mycelial extract of cultured Cordy ceps sinensis on in vivo hepatic energy metabolism and blood flow in dietary hypoferric anemic mice."British Journal of Nutrition. 83・2. 197-204 (2000)

[Publications] Noboru Manabe,Akira Myoumoto,Chiemi Tajima,Hajime Miyamoto et al.: "Immunochemical characteristics of a novel cell death receptor and a decoy receptor on granulosa cells of porcine ovarian follicles."Cytotechnology. 33・2. 189-201 (2000)

[Publications] Mizuho Nakayama,Noboru Manabe,Hajime Miyamoto et al.: "Species specific differences in apoptotic cell localization in granulosa and theca interna cells during follicular atresia in porcine and bovine ovaries."Journal of Reproduction and Development. 46・2. 147-156 (2000)

[Publications] Susumu Nishihara,Noboru Manabe,Hajime Miyamoto et al.: "Changes in cell adhesion molecules during follicular atresia in porcine ovaries."Journal of Reproduction and Development. 46・3. 325-334 (2000)

[Publications] Mizuho Nakayama,Noboru Manabe,Hajime Miyamoto et al.: "Changes in localization of type I, III and IV collagens and their mRNA expression during follicular atresia in bovine and porcine ovaries."Journal of Reproduction and Development. 47・1. 27-36 (2001)

[Publications] Kozue Uchio,Noboru Manabe,Kotaro Tamura,Makoto Miyamoto et al.: "Decreased matrix metalloproteinase activity in the kidneys of hereditary nephrotic mice (ICGN strain)."Nephron. 86・1. 145-141 (2000)

[Publications] Kotaro Tamura,Noboru Manabe,Kozue Uchio,Makoto Miyamoto et al.: "Characteristic changes in carbohydrate profile in the kidneys of hereditary nephrotic mice (ICGN strain)."The Journal of Veterinary Medical Science. 52・2. 379-390 (2000)

[Publications] Noboru Manabe,Akiko Kinoshita,Kozue Yamada-Uchio et al.: "Changes in quantitative profile of extracellular matrix components in the kidneys of rats with adriamycin-induced nephropathy."The Journal of Veterinary Medical Science. 53・1. 125-133 (2001)

[Publications] Miki Sugimoto,Noboru Manabe,Hajime Miyamoto.: "Future functional-morphological studies in embryology."Journal of Reproduction and Development. 46・1. 71-77 (2000)

[Publications] Noboru Manabe,Hajime Miyamoto et al.: "Immunohistochemical quantification of fast-myosin in upper hind limb muscles of the pig."Animal Science Journal. 72・2. 154-163 (2000)

[Publications] Miki Sugimoto,Noboru Manabe,Hajime Miyamoto.: "Development of infantile rat ovaries autotransplanted after cryopreservation by vitrification."Theriogenology. 53・11. 1093-1103 (2000)

[Publications] Noboru Manabe,Akira Myoumoto,Chiemi Tajima,Hajime Miyamoto et al.: "A novel cell death receptor and a decoy receptor on granulosa cells of porcine ovarian follicles."Tohoku University Press, Sendai, Japan.. 357 (2000)

宮本元京都大学, 農学研究科, 教授 (00026618)