HGFによる形態形成および器官形成・再生の分子生物的解析

1999 Fiscal Year Annual Research Report

• Project/Area Number: 11308025
• Research Category: Grant-in-Aid for Scientific Research (A)
• Research Institution: Osaka University
• Principal Investigator: 中村 敏一 大阪大学, 医学系研究科, 教授 (00049397)
• Co-Investigator(Kenkyū-buntansha): 船越 洋 大阪大学, 医学系研究科, 助手 (40273685) ; 小清水 右一 大阪大学, 医学系研究科, 助手 (50281126) ; 松本 邦夫 大阪大学, 医学系研究科, 助教授 (90201780)
• Keywords: HGF / c-met / トランスジェニックマウス / 神経 / HGFアンタゴニスト / 筋萎縮性側索硬化症 / ノックアウトマウス / ES細胞

Research Abstract

1.神経特異的HGF発現動物の作製とその解析
成体神経組織に特異的な発現を呈するneuron-specific enolaseプロモーターの下流にHGF cDNAを連結し、トランスジェニック(Tg)マウスの作製を行った。 遺伝子導入が確認できた9系統中、6系統で神経特異的なHGF発現・産生が認められた。 胎児および成体の脳・神経組織には特に異常はみられなかった。 現在、HGFの神経栄養因子としての機能を検討するため、これらのマウスの筋萎縮性側索硬化症(ALS)モデルのSOD-1 Tgマウスと交配し、神経の組織形態および機能に及ぼす影響を経時的に解析している。
2.ケラチノサイト特異的HGF発現動物の作製とその解析
ケラチン14プロモーターの下流でHGFを高発現するTgマウスを作製した。 このTgマウスでは皮膚や毛には特に異常を認められなかったが、メラノサイトとの異所性増殖がみられた。 胎児および成体皮膚組織の詳細な解析により、HGFはメラノサイトの遊走および増殖を促進することが明らかになった。
3.NK4発現動物の作製
HGFの分子内断面はNK4はHGFアンタゴニストとしての活性を有している。 メタロチオネインプロモーターの下流でNK4を発現するTgマウスの作製を行った。 遺伝子導入が確認できた15系統中、10系統でZn^<2+>投与によるNK4の高い発現誘導がみられた。 その主な産生臓器は肝臓、腎臓、小腸であり、系統によっては筋や脳、肺でも高発現がみられた。 現在、各種疾患モデル実験系を用いてNK4の器官・組織構築および修復に及ぼす影響を検討している。
4.C-metノックアウトマウスの作製
Cre-LoxPシステムを用いたc-metノックアウトマウスを作製するため、c-metゲノム遺伝子のクローニングを行い、キナーゼ領域の近傍(エクソン14〜18)を含むゲノム断片を基にターゲティングベクターを構築した。現在、このベクターをES細胞に導入して相同組み換え体の作製を進めている。

Research Products

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• [Publications] K.Kitajima,U.Koshimizu and T.Nakamura: "Expression of a novel type of classic cadherin, PB-cadherin in developing brain and limb buds"Develop.Dyn.. 215. 206 (1999)
• [Publications] N.Taniura-Matsumoto,K.Matsumoto and T.Nakamura: "Prostaglandin production in mouse mammary carcinoma cells confers invasive growth potential by inducing hepatocyte growth factor in fibroblasts"Brit.J.Cancer. 81. 194-202 (1999)
• [Publications] M.Kato,Y.Kato,T.Nakamura and Y.Sugiyama: "Efficient extraction by the liver governs the overall elimination of hepatocyte growth factor in rats"J.Pharmacol.Exp.Therap.. 290. 373-379 (1999)
• [Publications] Morishita,et al.: "Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischemia model as a cytokine supplement therapy"Hypertention. 33. 1379-1384 (1999)
• [Publications] W.G.Jiang,et al.: "Hepatocyte growth factor/scatter factor decreases the ixpression of occuludin and transendothelial resistance(TER) and increased paracellular permeability in human vascular endothelial cells"J.Cell.Physiol.. 181. 319-329 (1999)
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• [Publications] S.Deleu,et al.: "Immediate early gene expression in dog thyrocytes in response to growth proliferation and differentiation stimuli"J.Cell.Physiol.. 181. 342-354 (1999)
• [Publications] S.Deleu,et al.: "IGF-1 or insulin,and the TSH cyclic AMP cascade separately control dog and human thyroid cell growth and DNA synthesis, and complement each other in inducing mitogenesis"Mole.Cell.Endocrin.. 149. 41-51 (1999)
• [Publications] S.Hayashi,et al.: "Potential role of hepatocyte growth factor, a novel angiogenic growth factor in peripheral arterial disease : Downregulation of HGF in response to hypoxia in vascular cells"Circulation. 100. II301-II308 (1999)
• [Publications] T.Nagahori,et al.: "Interferon-gamma upregulates the c-Met/HGF receptor expression in alvedar epithelial cells"Am.J.Respir.Cell Mol.Biol.. 21. 490-497 (1999)
• [Publications] O.Amano,U.Koshimizu,T.Nakamura and S.Iseki: "Enhancement by hepatocyte growth factor (HGF) of bone and cartilage formation during embryonic mouse mandibular development in vitro"Arch.Oral Biol.. 44. 935-946 (1999)
• [Publications] K.Nishimura,et al.: "Prostate stromal cell-derived hepatocyte growth factor induces invasion of prostate cancer cellls DU 145 through Tumor-stromal interaction"Prostate. 41. 145-153 (1999)
• [Publications] W.Sun,H.Funakoshi and T.Nakamura: "Differential expression of hepatocyte growth factor and its receptor, c-Met in the rat retina during development"Brain Res.. 851. 46-53 (1999)
• [Publications] A.Uchiyama,et al.: "Hepatocyte growth factor and invasion-stimulatory activity are induced in pleural fluid by surgery in lung cancer patients"Br.J.Cancer. 81. 721-726 (1999)
• [Publications] K.Matsumoto,et al.: "Prevention of renal damage by Angiotensin II blockade, accompanied by increased renal hepatocyte growth factor in experimental hypertensive rats"Hypertension. 34. 279-284 (1999)
• [Publications] Y.Okura,et al.: "Analysis of neurotrophic factor of hepatocyte growth factor (HGF) in the adult hypoglossal nerve axotomy model"Eur J.Neurosci.. 11. 4139-4144 (1999)
• [Publications] T.Sumi,K.Matsumoto,Y.Takai and T.Nakamura: "Cofilin phosphorylation and actin cytoskeletal dynamics regulated by Rho-and Cdc42-activated LIM-kinase2"J.Cell Biol.. 147. 1519-1532 (1999)
• [Publications] MK.Jones,et al.: "HGF triggers activation of Cox-2 gene in rat gastric epithelial cells Action mediated through the ERK2 signalling pathway"FASEB J.. 13. 2186-2194 (1999)
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• [Publications] F.Tamura,et al.: "FK506 promotes liver regeneration by suppressing natural killer cell activity"J.Gastroenterol.and Hepatol.. (in press). (2000)
• [Publications] A.Uchiyama,et al.: "Significance of locally-secreted hepatocyte growth factor after thoracic cancer surgery as a stimulator for postoperative cancer invasion."Jpn.J.Clin.Oncol.. (in press). (2000)
• [Publications] W.G.Jiang,et al.: "Antagonistic effect of NK4,a novel HGF variant, on in vitro angiogenesis of human vascular endothelial cells"Clin.Cancer Res.. (in press). (2000)
• [Publications] S.Hiscox,et al.: "Inhibition of HGF/SF-induced breast cancer cell matility and invasion by the HGF/SF variant, NK4."Breast Cancer Res.& Treat.. (in press). (2000)
• [Publications] M.Nakano,et al.: "Hepatocyte growth factor is essential for amelioration of hyperglycemia in strepto zotocin-induced diabetic mice receiving a marginal mass of intrahepatic islet grafts"Transplantation. (in press). (2000)
• [Publications] T.Yamada,et al.: "Enhanced expression of hepatocyte growth factor by pulmonary ischemia reperfusion injury in the rat"Am.J.Resp.Crit Care Med.. (in press). (2000)
• [Publications] S.Mizuno,et al.: "Reciprocal balance between HGF and TGF-β a key determinant for pathological phenotypes in chronic renal disease"Kidney Intern.. (in press). (2000)
• [Publications] C.Parr,et al.: "NK4, a new HGF/SF variant,is an antagonist to the influence of HGF/SF on the matility and invasion of colon cancer cells"Intern.J.Cancer. (in press). (2000)
• [Publications] 中村敏一: "新しい腫瘍血管新生抑制物質-malignostatin(NK4)."細胞工学. 18. 223-225 (1999)
• [Publications] 久場敬司、松本邦夫、中村敏一: "HGFによる血管新生とその分子内クリングル断片(NK4)による腫瘍血管新生阻害"実験医学. 17. 734-740 (1999)
• [Publications] 水野信哉、中村敏一: "HGFによる急性および慢性腎不全の治療"腎と透析. 46. 494-498 (1999)
• [Publications] 久場敬司、中村敏一: "HGFと癌の浸潤・転移"Surgery Frontier 6(No2):27-38. 6. 27-38 (1999)
• [Publications] 小清水右一、中村敏一: "HGF-肝細胞癌に対するHGFの抗腫瘍作用"血液・免疫・腫瘍. 4. 40-45 (1999)
• [Publications] 中村隆弘、中村敏一: "肝再生の分子機序"別冊:医学のあゆみ、消化器疾患. 2. 68-73 (1999)
• [Publications] 久場敬司、中村敏一: "医学研究その15:血管新生(Angiogenesis)"The Lung. 7. 94-99 (1999)
• [Publications] 水野信哉、中村敏一: "肝細胞増殖因子による腎再生"最新医学. 54. 59-69 (1999)
• [Publications] 角 智行、中村敏一: "細胞遊走における増殖因子の役割"医学のあゆみ. 191. 137-142 (1999)
• [Publications] 水野信哉、中村敏一: "肝再生とHGF"現代医療. 31. 69-78 (1999)
• [Publications] 松本邦夫、中村敏一: "HGFの臨床応用に向けて"Medical Academy News. 720/721. 12-12 (1999)
• [Publications] 船越 洋、中村敏一: "肝細胞増殖因子(HGF)の検査値と各種疾患"日本臨牀. 37. 821-826 (1999)
• [Publications] 小清水右一中村敏一: "形質転換成長因子(HGF)"日本 臨牀. 37. 802-804 (1999)
• [Publications] 小清水右一、大熊勝治: "肝細胞の培養法"細胞生物学実験法. 1. 27-47 (1999)
• [Publications] 中村敏一、小池克郎、関谷剛男、近藤寿人: "タンパク質の分析法"分子生物学プロトコール 改訂2版. 11章. 269-284 (1999)
• [Publications] 中村敏一、長澤俊彦: "HGFによる腎疾患の治療"Annual Review 腎臓 2000. XII. 233-240 (2000)