2003 Fiscal Year Annual Research Report

内皮・平滑筋・繊維芽細胞の三次元共培養による血管構築を摸した血管モデルの作製

Research Project

Project/Area Number	14657174
Research Institution	Kyushu University
Principal Investigator	金出英夫九州大学, 大学院・医学研究院, 教授 (80038851)
Co-Investigator(Kenkyū-buntansha)	平野勝也九州大学, 大学院・医学研究院, 講師 (80291516) 西村淳二九州大学, 大学院・医学研究院, 助教授 (90237727)
Keywords	線維芽細胞 / 血管モデル / NIH3T3細胞 / Rho-kinase / Protein inase C / CPI-17 / GF10920X / 血管内皮細胞
Research Abstract	動脈硬化や血管形成術後の血管局所では、収縮性の亢進・攣縮を認めることがある。病変局所の平滑筋細胞や浸潤・増殖細胞において、収縮性亢進に関係する種々の酵素・蛋白質の発現変化が起こっている、と一般に考えられているが、その直接証拠は少ない。人工血管を作製し、これらの酵素・蛋白質の遺伝子を人工血管壁構成細胞に発現させることによって、収縮性の亢進・攣縮におけるその役割の直接証明が可能となる筈である。本年度は線維芽細胞のcell lineであるNIH3T3細胞を用いる事によって、等尺性収縮を測定可能な人工血管中膜モデルを作成した。さらにNIH3T3細胞に高効率に遺伝子導入できるベクターを開発し、CPI-17の過剰発現による線維芽細胞の収縮性の変化を検討した。CPI-17を過剰発現させた細胞とコラーゲンゲルを用いて作製した血管中膜モデルのリング標本は、PKCの活性化剤であるPDBuの投与により、10%FBSによる収縮の2-3倍の張力を発生した。一方、ベクターだけを発現させたコントロールではPDBuの投与により、むしろ基線以下に弛緩した。また、PKCによるリン酸化部位である38番目のThrをAlaあるはGluに変異させたCPI(CPI38AおよびCPI38E)では、PDBuによる収縮は得られず、コントロールと同様に弛緩した。CPI-17を過剰発現させた細胞のPDBuによる収縮は、PKC inhibitorであるGF109203Xにより抑制されたが、Rho kinase inhibtorであるY27632では抑制されなかった。線維芽細胞を中膜構成細胞として作製した人工血管モデルにおいては、PKCの活性化は本来は弛緩を起こすが、CPI-17を発現させると収縮に転じることが明らかになった。

Research Products

(12 results)

All Other

All Publications (12 results)

[Publications] Nakayama T: "Unproductive cleavage and inactivation of protease-activated receptor-1 by trypsin in vascular endotheial cells."Br J Pharmacol. 138. 121-130 (2003)
[Publications] Fukuyama K: "Down regulation of vascular angiorensin II type 1 receptor by thyroid hormone."Hypertension. 41. 598-603 (2003)
[Publications] Takahashi R: "The modulation of Ca^<2+> sensitivity regulates contractility of rabbit corpus cavernosum smooth muscle."J Urology. 169. 2412-2416 (2003)
[Publications] Hirano K: "Sequence requirement for nuclear localization and growth inhibition of p27^<Kip1R>, a degradation-resistant isoform of p27^<KiP1>."J Cell Biochem. 89. 191-202 (2003)
[Publications] Yamanaka J: "An important role for the Na^<+>-Ca^<2+> exchanger in the decrease in cytosolic Ca^<2+> concentration induced by isoprenaline in the porcine coronary artery."J Physiol. 549. 553-562 (2003)
[Publications] Eto W: "Intracellular alkalinization induces Ca^<2+> influx via non-voltage-operated Ca^<2+> channels in the rat aortic smooth muscle cells."Cell Calcium 34 : 477-484, 2003. 34. 477-484 (2003)
[Publications] Maeda Y: "Rho-kinase inhibitor inhibits both myosin phosphorylation-dependent and -independent enhancement of myofilament Ca^<2+> sensitivity in bovine middle cerebral artery."Br J Pharmacol. 140. 871-880 (2003)
[Publications] Ise S: "Theophylline attenuates Ca^<2+> sensitivity and modulates BK channels in porcine tracheal smooth muscle."Br J Pharmacol. 140. 939-947 (2003)
[Publications] Kanaide H: "Cellular mechanism of vasoconstriction induced by angiotensin II : It remains to be determined."Cir Res. 93. 1015-1017 (2003)
[Publications] Hirano K: "Transduction of the N-terminal fragments of MYPT1 enhances myofilament Ca^<2+> sensitivity in an intact coronary artery."Athersclerosis Thromb Vasc Biol. (in press).
[Publications] Nakayama T: "Inactivation of protease activated receptor-1 due to a proteolytic removal of the tethered ilgand by thrombin and trypsin in the human umbilical vein endothelial cells."Biochem Pharmacol. (in press).
[Publications] Koga M: "The long-term deficiency of estrogen enhances the contractile response without affecting the Ca^<2+> - sensitivity of the contractile apparatus in arterial smooth muscle of the female rabbit."J Soc Gynecol Invest. (in press).

2003 Fiscal Year Annual Research Report

内皮・平滑筋・繊維芽細胞の三次元共培養による血管構築を摸した血管モデルの作製

Principal Investigator

金出 英夫 九州大学, 大学院・医学研究院, 教授 (80038851)

Research Products

[Publications] Nakayama T: "Unproductive cleavage and inactivation of protease-activated receptor-1 by trypsin in vascular endotheial cells."Br J Pharmacol. 138. 121-130 (2003)

[Publications] Fukuyama K: "Down regulation of vascular angiorensin II type 1 receptor by thyroid hormone."Hypertension. 41. 598-603 (2003)

[Publications] Takahashi R: "The modulation of Ca^<2+> sensitivity regulates contractility of rabbit corpus cavernosum smooth muscle."J Urology. 169. 2412-2416 (2003)

[Publications] Hirano K: "Sequence requirement for nuclear localization and growth inhibition of p27^<Kip1R>, a degradation-resistant isoform of p27^<KiP1>."J Cell Biochem. 89. 191-202 (2003)

[Publications] Yamanaka J: "An important role for the Na^<+>-Ca^<2+> exchanger in the decrease in cytosolic Ca^<2+> concentration induced by isoprenaline in the porcine coronary artery."J Physiol. 549. 553-562 (2003)

[Publications] Eto W: "Intracellular alkalinization induces Ca^<2+> influx via non-voltage-operated Ca^<2+> channels in the rat aortic smooth muscle cells."Cell Calcium 34 : 477-484, 2003. 34. 477-484 (2003)

[Publications] Maeda Y: "Rho-kinase inhibitor inhibits both myosin phosphorylation-dependent and -independent enhancement of myofilament Ca^<2+> sensitivity in bovine middle cerebral artery."Br J Pharmacol. 140. 871-880 (2003)

[Publications] Ise S: "Theophylline attenuates Ca^<2+> sensitivity and modulates BK channels in porcine tracheal smooth muscle."Br J Pharmacol. 140. 939-947 (2003)

[Publications] Kanaide H: "Cellular mechanism of vasoconstriction induced by angiotensin II : It remains to be determined."Cir Res. 93. 1015-1017 (2003)

[Publications] Hirano K: "Transduction of the N-terminal fragments of MYPT1 enhances myofilament Ca^<2+> sensitivity in an intact coronary artery."Athersclerosis Thromb Vasc Biol. (in press).

[Publications] Nakayama T: "Inactivation of protease activated receptor-1 due to a proteolytic removal of the tethered ilgand by thrombin and trypsin in the human umbilical vein endothelial cells."Biochem Pharmacol. (in press).

[Publications] Koga M: "The long-term deficiency of estrogen enhances the contractile response without affecting the Ca^<2+> - sensitivity of the contractile apparatus in arterial smooth muscle of the female rabbit."J Soc Gynecol Invest. (in press).

金出英夫九州大学, 大学院・医学研究院, 教授 (80038851)