1995 Fiscal Year Annual Research Report

「血管壁ナトリウム利尿ペプチド系」の臨床的意義に関する発生学的、分子生物学的研究

Research Project

Project/Area Number	07671131
Research Institution	Kyoto University
Principal Investigator	伊藤裕京都大学, 原子炉実験所, 助手 (40252457)
Keywords	ノックアウトマウス / 遺伝子クローニング / アデノウィルス / 遺伝子導入 / 遺伝子治療 / 細胞増殖 / 血管平滑筋細胞
Research Abstract	1)マウスCNP遺伝子クローニングとCNPノックアウトマウスの開発:BALB/cマウス遺伝子ライブラリー10^6クローンをラットCNPcDNAプローブを用いてスクリーニングした。これよりマウスCNP遺伝子を含む2.6kbのDNA断片のクローニングに成功した。マウスCNP遺伝子はともに少なくとも3つのエクソンと2つのイントロンから構成されていることが明らかとなった。マウスCNP遺伝子5′-隣接領域の近位部にはTATA box, cAMP応答因子様配列、SP1結合部(GC box)、逆向きCCAAT配列(Y box)が認められた。また、その上流に25回のCA反復配列(マイクロサテライト)が認められた。我々は既にTGF-β及びTNF-αにより内皮細胞におけるCNP遺伝子発現が著明に亢進することを報告してきたが、マウスCNP遺伝子の5′-隣接領域及び第1イントロンには各々TGF-β応答因子様配列、NF-κB結合部位が存在しその重要性が示唆された。PCR法による組換え近交系マウス間のマイクロサテライト多型の解析により、マウスCNP遺伝子は、Ren-1遺伝子と同じ第1染色体上のAcrgとInの間に存在することが明らかになった。更に、マウスCNP遺伝子の第1エクソンの部分をネオマイシン耐性遺伝子にて破壊し、HSV-TK遺伝子を連結したターゲッティングベクターを作製した。このターゲッティングベクターをマウス胚幹細胞(ES細胞)にエレクトロポレーション法にて導入し、ホジティブ・ネガティブ選択法にて組同組換え体を選択し、生殖キメラマウスの作製に成功した。現在、ホモ接合体(ノックアウトマウス)を開発中である。 2)アデノウィルスを用いたCNP遺伝子導入による血管平滑筋細胞増殖抑制の検討及び増殖性血管病変の遺伝子治療への応用:E1部分を除いたヒト5型アデノウィルスゲノムとCMVプロモーター及びヒトCNP cDNAを組み込んだシャトルベクターを293細胞内で相同組換えさせることにより、CNP遺伝子発現アデノウィルスベクター(Ad. CMV/CNP)の作成に成功した。ラット大動脈血管平滑筋細胞に対し、Ad. CMV/CNPを40moi(pfu/cell)となるように感染させた。細胞培養上清中のCNP濃度は、Ad. CMV/CNP感染群では122±44pmol/10^6 cellsであり、これは内皮細胞のCNP産生(32±5fmol/10^6 cells)の約4000倍であった。このAd. CMV/CNP感染群ではほぼ完全な増殖抑制効果が認められた。アデノウィルスベクターにより遺伝子導入されたCNPは血管平滑筋細胞に対してオートクリン、パラクリン因子として増殖抑制効果を発揮し、CNP遺伝子の過剰発現は増殖性血管病変に対する遺伝子治療に有用であることが示唆された。

Research Products

(22 results)

All Other

All Publications (22 results)

[Publications] O. Nakagawa et al.: "Rapid transcription activation and early mRNA turnover of brain natriuretic peptide in cardiocyte hypertrophy-Evidence for grain natriuretic peptide as an “emergency cardiac hormone against ventricular overload-." J. Clin. Invest.96. 1280-1287 (1995)
[Publications] Y. Ogawa et al.: "Structural organization and chromosomal assignment of the human prostacyclin receptor gene." Genomics. 27. 142-148 (995)
[Publications] S. Sugiyama et al.: "Lipoproteins regulate C-type natriuretic peptide secretion from cultured vascular endothelial cells." Arterioscler. Thromb. Vasc. Biol.15. 1968-1974 (1995)
[Publications] N. Hama et al.: "Rapid ventricular induction of brain natriuretic gene expression in experimental acute myocardial infarction." Circulation. 92. 1558-1564 (1995)
[Publications] J. Yamashita et al.: "Cis elements for transcriptional regulation of the human endothelin-A receptor gene." J. Cardiovasc. Pharmacol.26(Suppl. 3). S26-S28 (1995)
[Publications] Y. Miyamoto et al.: "Multiple transcripts of human endothelin-A receptor gene detected by reverse transcription and the polymerase chain reaction." J. Cardiovasc. Pharmacol.26(Suppl. 3). S29-S31 (1995)
[Publications] J. Hiraoka et al.: "Augmented expression of the endothelin-A receptor gene in cultured mesangial cells from stroke-prove spontaneously hypertensive rats." Clinical and Experimental Pharmacology and Physiology. Suppl 1. S191-S192 (1995)
[Publications] O. Nakagawa et al.: "Gene expression of prostacyclin receptor in the hypertrophied heart of spontaneously hypertensive rats." Clinical and Experimental Pharmacology and Physiology. Suppl 1. S270-S272 (1995)
[Publications] O. Nakagawa et al.: "Gene regulation of brain natriuretic peptide in cardiocyte hypertrophy by α_1-adrenergic stimulation" Clinical and Experimental Pharmacology and Physiology. Suppl 1. S183-S185 (1995)
[Publications] I. Kishimoto et al.: "Endothelin-induced activation of mitogen-activated protein kinases in glomerular mesargial cells from normotensive and stroke-prone spontaneously hypertensive rats." Clinical and Experimental Pharmacology and Physiology. Suppl 1. S207-S208 (1995)
[Publications] M. Nakagawa et al.: "Preparation of a monoclonal antibody against mouse brain natriuretic peptide(BNP) and tissue distribution of BNP mice." Clinical and Experimental Pharmacology and Physiology. Suppl. 1. S186-S187 (1995)
[Publications] M. Goto et al.: "Altered gene expression of natriuretic peptide receptor subtypes in the K idnoy of stroke-prove spontaneously hypertensive rats." Clinical and Experimental Pharmacology and Physiology. Suppl 1. S177-S179 (1995)
[Publications] Y. Ogawa et al.: "Molecular biology and biochemistry of natriuretic peptide family." Clinical and Experimental Pharmacology and Physiology. 22. 49-53 (1995)
[Publications] H. Itoh at al.: "Significance of autocrine/paracrine network in renal vascular and mesangial growth." Jpn. Heart J.36. 501- (1995)
[Publications] T. Ikeda et al.: "Natriuretic peptide receptors in human arterial and venous coronary bypass vessels and rabbit vern grafts." Hypertension. (in press).
[Publications] K. Doi et al.: "Vascular endothelial growth factor potently suppresses CNP secretion from endothelial cells." Hypertension. (in press).
[Publications] N. Tamura et al.: "cDNA cloning and gene expression of human type I alpha cGMP-dependent protein kinase." Hypertension. (in press).
[Publications] Y. Komatsu et al.: "Regulation of endothelial production of C-type natriuretic peptide in coculture with vascular smooth muscle cells-The role of vascular natriuretic peptide system in vascular growth inhibition." Circ. Res.(in press).
[Publications] Y. Miyamoto et al.: "Alternative RNA splicing of human endothelin-A receptor generates multiple transcriipts." Biochemical J.(in press).
[Publications] T. Igaki et al.: "C-type natriuretic peptide in chronic renal failure and its action in humans." Kidney Int.(in press).
[Publications] T. Igaki et al.: "Insulin suppresses endothelial secretion of C-type natriuretic peptide, a novel endothelium-derived relaxing peptide." Diabetes. (in press).
[Publications] Y. Komatsu et al.: "Regulation of secretion and clearance of C-type natriuretic peptide in the interaction of vascular endothelial cells and smooth muscle cells." J. Hypertens.(in press).

1995 Fiscal Year Annual Research Report

「血管壁ナトリウム利尿ペプチド系」の臨床的意義に関する発生学的、分子生物学的研究

Principal Investigator

伊藤 裕 京都大学, 原子炉実験所, 助手 (40252457)

Research Products

[Publications] O. Nakagawa et al.: "Rapid transcription activation and early mRNA turnover of brain natriuretic peptide in cardiocyte hypertrophy-Evidence for grain natriuretic peptide as an “emergency cardiac hormone against ventricular overload-." J. Clin. Invest.96. 1280-1287 (1995)

[Publications] Y. Ogawa et al.: "Structural organization and chromosomal assignment of the human prostacyclin receptor gene." Genomics. 27. 142-148 (995)

[Publications] S. Sugiyama et al.: "Lipoproteins regulate C-type natriuretic peptide secretion from cultured vascular endothelial cells." Arterioscler. Thromb. Vasc. Biol.15. 1968-1974 (1995)

[Publications] N. Hama et al.: "Rapid ventricular induction of brain natriuretic gene expression in experimental acute myocardial infarction." Circulation. 92. 1558-1564 (1995)

[Publications] J. Yamashita et al.: "Cis elements for transcriptional regulation of the human endothelin-A receptor gene." J. Cardiovasc. Pharmacol.26(Suppl. 3). S26-S28 (1995)

[Publications] Y. Miyamoto et al.: "Multiple transcripts of human endothelin-A receptor gene detected by reverse transcription and the polymerase chain reaction." J. Cardiovasc. Pharmacol.26(Suppl. 3). S29-S31 (1995)

[Publications] J. Hiraoka et al.: "Augmented expression of the endothelin-A receptor gene in cultured mesangial cells from stroke-prove spontaneously hypertensive rats." Clinical and Experimental Pharmacology and Physiology. Suppl 1. S191-S192 (1995)

[Publications] O. Nakagawa et al.: "Gene expression of prostacyclin receptor in the hypertrophied heart of spontaneously hypertensive rats." Clinical and Experimental Pharmacology and Physiology. Suppl 1. S270-S272 (1995)

[Publications] O. Nakagawa et al.: "Gene regulation of brain natriuretic peptide in cardiocyte hypertrophy by α_1-adrenergic stimulation" Clinical and Experimental Pharmacology and Physiology. Suppl 1. S183-S185 (1995)

[Publications] I. Kishimoto et al.: "Endothelin-induced activation of mitogen-activated protein kinases in glomerular mesargial cells from normotensive and stroke-prone spontaneously hypertensive rats." Clinical and Experimental Pharmacology and Physiology. Suppl 1. S207-S208 (1995)

[Publications] M. Nakagawa et al.: "Preparation of a monoclonal antibody against mouse brain natriuretic peptide(BNP) and tissue distribution of BNP mice." Clinical and Experimental Pharmacology and Physiology. Suppl. 1. S186-S187 (1995)

[Publications] M. Goto et al.: "Altered gene expression of natriuretic peptide receptor subtypes in the K idnoy of stroke-prove spontaneously hypertensive rats." Clinical and Experimental Pharmacology and Physiology. Suppl 1. S177-S179 (1995)

[Publications] Y. Ogawa et al.: "Molecular biology and biochemistry of natriuretic peptide family." Clinical and Experimental Pharmacology and Physiology. 22. 49-53 (1995)

[Publications] H. Itoh at al.: "Significance of autocrine/paracrine network in renal vascular and mesangial growth." Jpn. Heart J.36. 501- (1995)

[Publications] T. Ikeda et al.: "Natriuretic peptide receptors in human arterial and venous coronary bypass vessels and rabbit vern grafts." Hypertension. (in press).

[Publications] K. Doi et al.: "Vascular endothelial growth factor potently suppresses CNP secretion from endothelial cells." Hypertension. (in press).

[Publications] N. Tamura et al.: "cDNA cloning and gene expression of human type I alpha cGMP-dependent protein kinase." Hypertension. (in press).

[Publications] Y. Komatsu et al.: "Regulation of endothelial production of C-type natriuretic peptide in coculture with vascular smooth muscle cells-The role of vascular natriuretic peptide system in vascular growth inhibition." Circ. Res.(in press).

[Publications] Y. Miyamoto et al.: "Alternative RNA splicing of human endothelin-A receptor generates multiple transcriipts." Biochemical J.(in press).

[Publications] T. Igaki et al.: "C-type natriuretic peptide in chronic renal failure and its action in humans." Kidney Int.(in press).

[Publications] T. Igaki et al.: "Insulin suppresses endothelial secretion of C-type natriuretic peptide, a novel endothelium-derived relaxing peptide." Diabetes. (in press).

[Publications] Y. Komatsu et al.: "Regulation of secretion and clearance of C-type natriuretic peptide in the interaction of vascular endothelial cells and smooth muscle cells." J. Hypertens.(in press).

伊藤裕京都大学, 原子炉実験所, 助手 (40252457)