フコース転移酵素アンチセンスオリゴヌクレオタイドを用いた肝細胞癌治療

• 研究課題/領域番号: 09670520
• 研究種目: 基盤研究(C)
• 配分区分: 補助金
• 応募区分: 一般
• 研究分野: 消化器内科学
• 研究機関: 新潟大学
• 研究代表者: 青柳 豊 新潟大学, 医学部, 助教授 (00142266)
• 研究分担者: 鈴木 健司 新潟大学, 医学部, 助手 (00303123) ; 大越 章吾 新潟大学, 医学部・附属病院, 助手 (70231199) ; 小谷 昌司 新潟大学, 理学部, 教授 (60018702) ; 鈴木 康史 新潟大学, 医学部, 助手 (50270939) ; 小方 則夫 新潟大学, 医学部・附属病院, 助手 (70204063)
• 研究期間 (年度): 1997 – 1999
• 研究課題ステータス: 完了 (1999年度)
• 配分額: 3,100千円 (直接経費: 3,100千円); 1999年度: 600千円 (直接経費: 600千円); 1998年度: 500千円 (直接経費: 500千円); 1997年度: 2,000千円 (直接経費: 2,000千円)
• キーワード: フコース転移酵素 / アンチセンス / 肝細胞癌 / 治療 / Lens culinaris agglutinin / treatment

研究概要

報告者らは肝細胞癌患者における各種血清糖蛋白においてフコシル化が亢進していることを報告し,この事象が腫瘍の悪性度の指標になることから,フコース転移酵素(α1-6FT)のmRNAに対するアンチセンスによる肝細胞癌治療の可能性に関する基礎的検討を行った.すなわち,5型グルコサミン転移酵素(Gnt-V)とα1-6FTに対するアンチセンスRNAを持続的に発現する系を確立しその生物学的特性の評価を試みた.テトラサイクリンの制御系に関してはClontech社(USA)のTet-off systemを用い,pTet-offベクターをHeLa細胞にトランスフェクトし,ネオマイシン耐性により持続安定発現株を選択,樹立した.次に,pTREベクターにNishikawa,A.ら(BBRC198,318-327,1994)によって報告されているGnt-VのmRNA配列と,Yanagidani,S.ら(J.Biochem.121,626-632,1997)によって報告されているα1-6FTのmRNAの配列に基づきプライマーを設定し,TR-PCRによってGnt-V(nt.54-495)とα1-6FT(nt.-20-693)のcDNAをHepG2より調製し,クローニングした.これらのクローニングされたベクターと,選択マーカーであるハイグロマイシン耐性遺伝子を持つpTK-Hygとをカチオニックリポソームを用いて,テトラサイクリンリプレッサー蛋白を持続発現しているHeLa細胞にコトランスフェクトし,ハイグロマイシンで選択した.約10^5のHeLa細胞を用いてリポフェクションを施行すると,50前後のハイグロマイシン耐性のクローンを選択する事が出来た.そして,やや不安定ながら,ドキシサイクリン存在下にセンス,あるいはアンチセンスRNAを発現する株を樹立し,現在その生物学的特性を評価中である.

研究成果

• [文献書誌] 馬場勝也: "The amino acid sequence of a lamprey(Entosphenus japonicus)liver fatty acid-binding protein identified its close relationship to cardiac fatty acid-binding proteins of Mammalia"Comparative Biochemistry and Physiology. 123. 223-228 (1999)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 五十川 修: "Reduction of telomeric repeat as a possible predictor for development of hepatocellular carcinoma ; convenient evaluation by slot-blot analysis"Hepatology. 30. 408-412 (1999)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 見田有作: "Plasma fucosyltransferase activity in patients with hepatocellular carcinoma, with special reference to correlation with fucosylated species of alpha-fetoprotein"J. Hepatology, 2000. (in press).
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 斉藤 崇: "Elevation of TFF1 (pS2) gene expression during healing of gastric ulcer at 'non-ulcerated' sites in the stomach : Semi-quantification using single tube method of polymerase chain reaction"J Gastroenterol Hepatol. 2000. (in press).
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 川合弘一: "Asymptomatic primary sclerosing cholangitis with marked hepatic fibroses"Digestive diseases & Science 2000. (in press).
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 滝沢英昭: "A case with diffuse narrowing of the main pancreatic duct successfully treated by steroid therapy"Digestive Endoscopy, 2000. (in press).
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 斉藤敦: "Measurement of the fucosylated sugar chains of serum alpha-1-antitrypsin : The relationship between reactivity with Lens culinaris agglutinin and HPLC analysis of the pyridylaminated sugar chains"Acta Medica et Biologica. 45・4. 11-14 (1997)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 曽我憲二: "Serum des-gamma-carboxy prothrombin level by a modified enzyme immunoassay method in hepatocellular carcinoma-Significance of des-gamma-carboxy prothrombin in small hepatocellular carcinoma"Hepato-Gastoenterology. 45. 1737-1741 (1998)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 須田剛士: "Quantitation of telomerase activity in hepatocellular carcinoma : A possible aid for a prediction of recurrent disease in the remnant liver"Hepatology. 27. 402-406 (1998)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 森茂 紀: "N-acetyl-glucosaminyltransferase III activities in hepatocellular carcinoma"J Gastroenterol Hepatol. 13. 610-619 (1998)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 見田 有作: "Usefulness of sensitive determination of des- γ -carboxy prothrombin in the early diagnosis of patients with hepatocellular carcinoma"Cancer. 82. 1643-1648 (1998)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 青柳豊: "Fucosylation index of alpha-fetoprotein as a possible prognostic indicator in patients with hepatocellular carcinoma"Cancer. 83. 2076-2082 (1998)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 内籐彰: "Highly enhanced fucosylation of serum glycoproteins in patients with hepatocellular carcinoma"J Gastroenterol Hepatol. 14. 432-441 (1999)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 大石健太郎: "Bilirubin binding activity of cytokeratin 1 isolated from the porcine liver"Cell Structure and Function. 23. 325-331 (1998)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 庄司豊: "Cell adhesion activity for murine carcinoma cells of a wheat germ 55kDa protein with binding affinity for animal extracellular matrix proteins"Biochim. biophys. Acta. 1426. 498-504 (1999)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 佐伯晃一: "Apoptosis-inducing activity of polyphenol compounds derived from tea catechins in human histiolytic lymphoma U937 cells"Biochim. Biophys. Acta. 63(3). 585-587 (1999)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 横田隆司: "The striking effect of hyperbaric oxygenation therapy in the management of chronic idiopathic intestinal pseudo-obstruction"Am J Gastroenterol. 95. 285-288 (2000)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 青柳 豊: "消化器疾患"医歯薬出版. 562 (1999)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 青柳 豊: "肝臓病"中外医学社. 318 (1997)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 青柳 豊: "Methods in Molecular Medicine, Lectin Methods and Protocols, lectins for detection of altered glycosylation of circulating glycoproteins : alpha-1-antitrypsin"Humana Press Inc. Totowa, NJ Rhodes, JM & Milton, JD. Eds.. 214 (1997)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] Saitoh A, Aoyagi Y, Mori S, Suda T, Suzuki Y, Sekine C and Asakura, H.: "Measurement of the fucosylated sugar chains of serum alpha-1-antitrypsin : The relationship between reactivity with Lens culinaris agglutinin and HPLC analysis of the pyridylaminated sugar chains."Acta Medica et Biologica. 45. 11-14 (1997)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Suda T, Isokawa O, Aoyagi Y, Nomoto M, Tsukada K, Shimizu T, Suzuki Y, Naitoh A, Igarashi H, Yanagi M, Takahashi T, Asakura H.: "Quantitation of telomerase activity in hepatocellular carcinoma : A possible aid for a prediction of recurrent disease in the remnant liver."Hepatology. 27. 402-406 (1998)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Mita Y, Aoyagi Y, Yanagi M, Suzuki Y, Asakura H.: "Usefulness of sensitive determination of des-gamma-carboxy prothrombin in the early diagnosis of patients with hepatocellular carcinoma"Cancer. 82. 1643-1648 (1998)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Mori S, Aoyagi Y, Yanagi M, Suzuki Y, Asakura H.: "N-acetyl-glucosaminyltransferase III activities in hepatocellular carcinoma."J Gastroenterol Hepatol.. 13. 610-619 (1998)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Soga K, Watanabe T, Aikawa K, Toshima M, Shibasaki K. and Aoyagi Y.: "Serum des-gamma-carboxy prothrombin level by a modified enzyme immunoassay method in hepatocellular carcinoma."Hepato-Gasroenterology. 45. 1737-1741 (1998)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Aoyagi Y, Isokawa O, Suda T, Watanabe M, Suzuki Y, Asakura H: "Fucosylation index of alpha-fetoprotein as a possible prognostic indicator in patients with hepatocellular carcinoma."Cancer. 83. 2076-2082 (1998)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Ohishi K, Mita T, Odani S, Takahashi T, Aoyagi Y, Munakata H, Tada K, Isemura S, Hsu CC, Isemura M.: "Bilirubin binding activity of cytokeratin 1 isolated from the porcine liver."Cell Structure and Function. 23. 325-331 (1998)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Takizawa H, Natsui M, Aoyagi Y, Asakura H.: "Pancreatitis in ulcerative colitis : A case report of improved stricture of the main pancreatic duct."J Clin Gastroenterology. 28. 74-76 (1999)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Saeki K, Sano M, Miyase T, Nakamura Y, Hara Y, Aoyagi Y, Isemura M.: "Apoptosis-inducing activity of polyphenol compounds derived from tea catechins in human histiolytic lymphoma U937 cells."Biosci. Biotechnol. Biochem.. 63(3). 585-587 (1999)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Naitoh A, Aoyagi Y, Asakura H.: "Highly enhanced fucosylation of serum glycoproteins in patients with hepatocellular carcinoma, correlation of the fucosylations berween alpha-fetoprotein, alpha-1-antitrypsin and transferrin."J Gastroenterol Hepatol.. 14. 432-441 (1999)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Shoji Y, Aoyagi Y, Kasakami T, Isemura S, Isemura M.: "Cell adhesion activity for murine carcinoma cells of a wheat germ 55kDa protein with binding affinity for animal extracellular matrix proteins."Biochim. Biophys. Acta,. 1426. 498-504 (1999)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Suda T, Aoyagi Y.: "A pregnant case of severe acute hepatitis type C : Successfully treated with natural interferon alpha and no evidence of maternal transmission of HCV to the new born."Acta Medica et Biologica,. 47. 75-80 (1999)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Baba K, Takahashi Y, Aoyagi Y, Odani, S.: "The amino acid sequence of a lamprey (Entosphenus japonicus) liver fatty acid-binding protein identified its close relationship to cardiac fatty acid-bindingproteins of Mammalia."Comparative Biochemistry and Physiology. 123. 223-228 (1999)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Isokawa O, Suda T, Aoyagi Y, Kawai H, Takahashi T, Tsukada K, Shimizu T, Nomoto M, Igarashi H, Yanai M, Suzuki Y, Asakura H.: "Reduction of telomeric repeat as a possible predictor for development of hepatocellular carcinoma ; convenient evaluation by slot-blot analysis."Hepatology. 30. 408-412 (1999)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Furukawa K, Yokoyama A, Sato K, Horikawa K, Kurita Y, Aoyagi Y.: "Aberrant systemic arterial supply to the non-sequestrated lung : A case repor."Acta Medica et Biologica. 47. 121-124 (1999)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Yokota T, Suda T, Tsukioka S, Takahashi T, Honma T, Seki K, Matsuzawa J, Miura M, Aoyagi Y, Asakura H.: "The striking effect of hyperbaric oxygenation therapy in the management of chronic idiopathic intestinal pseudo-obstruction."Am J Gastroenterol. (in press.). (1999)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Mita Y, Aoyagi Y, Suda T, Asakura H,: "Plasma fucosyltransferase activity in patients with hepatocellular carcinoma, with special reference to correlation with fucosylated species of alpha-fetoprotein."J Hepatology. (in press.). (2000)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Kawai H, Aoyagi Y, Nomoto M, Takizawa H, Suzuki Y, Hama A, Suda T, Takahashi T, Asakura H,: "Asymptomatic primary sclerosing cholangitis with marked hepatic fibrosis."Digestive Diseases & Science. (in press.). (2000)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Takizawa H, Suzuki Y, Aoyagi Y, Asakura H.: "A case with diffuse narrowing of the main pancreatic duct successfully treated by steroid therapy."Digestive Endoscopy. (in press.). (2000)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Saitoh T, Mochizuki T, Suda T, Aoyagi Y, Tsukada Y, Narisawa N, Asakura H,: "Elevation of TFF1 (pS2) gene expression during healing of gastric ulcer at 'non-ulcerated' sites in the stomach : Semi-quantification using single tube method of polymerase chain reaction."J Gastroenterol Hepatol. (in press.). (2000)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] 馬場 勝也: "The amino acid sequence of a lamprey (Entosphenus japonicus) liver fatty acid-binding protein identified its close relationship to cardiac fatty acid-binding proteins of Mammalia."Comparative Biochemistry and Physiology,. 123. 223-228 (1999)
• [文献書誌] 五十川 修: "Reduction of telomeric repeat as a possible predictor for development of hepatocellular carcinoma; convenient evaluation by slot-blot analysis."Hepatology,. 30. 408-412 (1999)
• [文献書誌] 見田 有作: "Plasma fucosyltransferase activity in patients with hepatocellular carcinoma, with special reference to correlation with fucosylated species of alpha-fetoprotein."J Hepatology, 2000. (in press). (2000)
• [文献書誌] 斉藤 崇: "Elevation of TFF1 (pS2) gene expression during healing of gastric ulcer at 'non-ulcerated' sites in the stomach: Semi-quantification using single tube method of polymerase chain reaction."J Gastroenterol Hepatol, 2000. (in press). (2000)
• [文献書誌] 川合 弘一: "Asymptomatic primary sclerosing cholangitis with marked hepatic fibrosis."Digestive Diseases & Science 2000. (in press). (2000)
• [文献書誌] 滝沢 英昭: "A case with diffuse narrowing of the main pancreatic duct successfully treated by steroid therapy."Digestive Endoscopy, 2000. (in press). (2000)
• [文献書誌] 青柳 豊: "消火器疾患"医歯薬出版. 562 (1999)
• [文献書誌] 青柳 豊: "肝臓病"中外医学社. 318 (1997)
• [文献書誌] 青柳豊: "Fucosylation index of alpha-fetoprotein as a possible prognostic indicator in patients with hepatocellular carcinoma." Cancer. 83. 2076-2082 (1998)
• [文献書誌] 内藤彰: "Highly enhanced fucosylation of serum glycoproteins in patients with hepatocellular carcinoma." J Gastroenterol Hepatol. 14. 432-441 (1999)
• [文献書誌] 大石健太郎: "Bilirubin binding activity of cytokeratin 1 isolated from the porcine liver." Cell Structure and Function. (in press). (1999)
• [文献書誌] 庄司 豊: "Cell adhesion activity for murine carcinoma cells of a wheat germ 55kDa protein with binding affinity for animal extracellular matrix proteins." Biochim.Biophys.Acta. (in press). (1999)
• [文献書誌] 佐伯晃一: "Apoptosis-inducing activity of polyphenol compounds derived from tea catechins in human histiolytic lymphoma U937 cells." Biochim.Biophys.Acta. 63(3). 585-587 (1999)
• [文献書誌] 横田 隆司: "The striking effect of hyperbaric oxygenation therapy in the management of chronic idiopathic intestinal pseudo-obstuction." Am J Gastroenterol. (in press). (1999)
• [文献書誌] 斉藤敦: "Measurement of the fucosylated sugar chains of serum α-1-antitrypsin : The relationship between reactivity with Lens culinaris agglutinin and HPLC analysis of the pyridylaminated sugar chains" Acta Medica et Biologica,. 45・4. 11-14 (1997)
• [文献書誌] 曽我憲二: "Scrum des-γ-carboxy prothrombin level by a modified enzyme immunoassay method in hepatocel-lular carcinoma-Significance of des-γ-carboxy prothrombin in small hepatocellular carcinoma" Hepato-Gasroenterology. (in press). (1998)
• [文献書誌] 須田剛士: "Quantitation of telomerase activity in hepatocellular carcinoma : A possible aid for a prediction of recurrent disease in the remnant liver" Hepatology. 27. 402-406 (1998)
• [文献書誌] 森茂紀: "N-acetyl-glucosaminyltransferase III activities in hepatocellular carcinoma." J Gastroenterol Hepatol.(in press). (1998)
• [文献書誌] 見田有作: "Usefulness of sensitive determination of des-γ-carboxy prothrombin in the early diagnosis of patients with hepatocellular carcinoma" Cancer. (in press). (1998)
• [文献書誌] 青柳豊: "Methods in Molecular Medicine,Lectin Methods and Protocols,Lectins for detection of altered glycosylation of circulating glycoproteins : α-l-antitrypsin" Humana Press Inc.Totowa,NJ Rhodes,JM & Milton,JD.Eds., 207-214 (1997)