血栓、止血障害の遺伝子治療を目指した分子生物学的解析

1993 Fiscal Year Annual Research Report

• Project/Area Number: 05304036
• Research Institution: Hiroshima University
• Principal Investigator: 藏本 淳 広島大学, 原爆放射能医学研究所, 教授 (50034070)
• Co-Investigator(Kenkyū-buntansha): 斉藤 英彦 名古屋大学, 医学部, 教授 (20153819) ; 大熊 稔 京都大学, 医学部, 教授 (50026986) ; 岩永 貞昭 九州大学, 理学部, 教授 (90029942) ; 千谷 晃一 藤田保健衛生大学, 総合医科学研究所, 教授 (60179942) ; 青木 延雄 東京医科歯科大学, 医学部, 教授 (20048937)
• Keywords: 凝固線溶調節因子欠損症 / エンドトキシンショック / 血小板膜レセプター異常症 / レセプター機能ドメイン / vWF活性化因子 / 遺伝子組み換え技術 / 遺伝子解析 / アンチセンス療法

Research Abstract

本研究班は血栓形成にかかわり合う種々の因子の確定と調節の分子機構を遺伝子レベルで明らかにし、血栓症の発症、再発予防及び血栓症そのものの遺伝子治療を目指すものである。
1.凝固調節因子の中でProtein S(PS)やProtein C(PC)について検討しPS欠損症2家系での遺伝子解析を行い一例はエクソン13のArg474→Cysのミスセンスミューテーション、二例目はエクソン14のGln522→stopのノンセンスミューテーションであった。またPC欠損症の遺伝子治療のためにPC cDNAをLNCXおよびLXSNベクターに挿入し、PC cDNA組み込みウイルス粒子を作成した。
2.凝固因子については血友病Bの遺伝子治療の前段階としてヒトIX因子の糖鎖構造を解析しアクチベーションペプタイドにはO-結合型糖鎖を含むものと含まないものが存在することを明らかにした。
3.線溶系ではウロキナーゼレセプターをCHO細胞に遺伝子工学的に発現させ、これを抗原としてモノクロナール抗体を作成した。線溶系の制御に必要なα2プラスミンインヒビター欠損症において第7エクソンの137番目にグルタミン酸の欠失のためα2プラスミンインヒビターが分泌されず細胞内に蓄積していることが判った。
4.エンドトキシンショックには多くのサイトカインがその発症に関係している。エンドトキシンやTNFによって活性化される核内転写活性化因子NF-kBを抑えるアンチセンスNF-kB ODNを用いたところ、IL-6、MHC class I,TNF-αの発現が抑制され死亡率が減少した。
5.in vivo vWF活性化因子が血管内皮細胞表面の45KD蛋白にフィブロネクチンが結合することによって内皮細胞上に形成されることを示した。
6.血小板のコラーゲン粘着反応を阻害する成分(HRIA)を蛇毒の中から見出し膜蛋白を特異的に分解している可能性を示した。強力な血小板活性化物質であるトロンボキサンA2(TXA2)に対するレセプター異常症(TXA2受容体異常症)の遺伝子解析を行い細胞質内第一ループにArg→Leuのミスセンス変異を示しGTP結合蛋白との関連の障害を推測した。血小板凝集反応を抑制し抗血栓薬の開発を導くためフィブリノーゲンレセプター(αIIbβ3)の機能ドメインをDNA組み換え技術を用いて検討しαIIbのN末領域にαIIbβ3のレセプター特異的機能のあることを示した。

Research Products

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• [Publications] Tohru Miki: "Molecular cloning of the breakpoint for 3q27 translocation in B-cell lymphomas and leukemias." Blood. 83. 217-222 (1994)
• [Publications] Tohru Miki: "Gene involved in the 3q27 translocation associated with B-cell lymphoma,BCL-5,encodes a Kruppel-like zinc-finger protein." Blood. 83. 26-32 (1994)
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• [Publications] Y.Usami: "Primary Structure of Two-chain Botrocetin,a von Willebrand Factor Modulator Purified from Venom of Bothrops jararaca" Proc.Natl.Acad.Sci.USA. 90. 928-932 (1993)
• [Publications] E.Yoshida: "Alboaggregin-B and Botrocetin,Two Snake Venom Proteins,with Highly Homologous Amino Acid Sequences but Totally Distinct---" Biochem.Biophys.Res.Commun.191. 1386-1392 (1993)
• [Publications] T.Yamada: "Structural and Functional Analyses of the Arg-Gly-Asp Sequence Introduced into Human Lysozyme" J.Biol.Chem.268. 10588-10592 (1993)
• [Publications] T.Matsui: "Human Plasma α2-Macroglobulin and von Willebrand Factor Posses Covalently-linked ABO(H)Blood Group Antigens in---" Blood. 82. 663-668 (1993)
• [Publications] Fuminori Tokunaga: "Limulus Hemocyte Transglutaminase:Its Purification and Characterization,and Identification of the Intracellular Substrates." J.Biol.Chem.268. 252-261 (1993)
• [Publications] Fuminori Tokunaga: "Limulus Hemocyte Transglutaminase:cDNA Cloning,Amino Acid Sequence,and Tissue Localization." J.Biol.Chem.268. 262-268 (1993)
• [Publications] Hitoshi Nishimura: "Factor IX Fukuoka:Substitution of Asn-92 by His in the Second Epidermal Growth Factor-like Domain Results in Defective Interaction with Factors VIII/X." J.Biol.Chem.268(32). 24041-24046 (1993)
• [Publications] Tatsushi Muta: "Horseshoe Crab Coagulation Factor B:A Unique Serine Protease Zymogen Activated by a Cleavage between Ile-Ile Bond." J.Biol.Chem.268. 21384-21388 (1993)
• [Publications] Shun-Ichiro Kawabata: "Rabbit Liver Microsomal Endopeptidase with Substrate Specificity for processing proproteins is Structurally Related to Rat Tests Metalloendopeptidase 24.15." J.Biol.Chem.268(17). 12498-12503 (1993)
• [Publications] Nahoki Kuraya: "Structural Analysis of 0-linked Sugar Chains in Human Blood Clotting Factor IX." J.Biochem.114(6). 763-765 (1993)
• [Publications] K.Hashino: "Engineering of Cell Adhesive Immunoglobulin by Grafting the Arg-Gly-Asp Cell Adhesive Signal" Biosci.Biotech.Biochem.58. 191-192 (1994)
• [Publications] Matsuda,S.: "Decreased messenger RNA of arachidonate 12-lipoxygenase in platelet of patients with myeloproliferative disorders." Biochim Biophys Acta. 1180. 243-249 (1993)
• [Publications] Fuse,I.: "Defective signal transduction induced by thromboxane A2 in a patient a mild bleeding disorder:lmpaired phospholipase C activation despite normal phosphlolipase A activation." Blood. 81. 994-1000 (1993)
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• [Publications] Hidehiko Saito: "A phenotypically neutral dimorphism of protein S:the substitution of Lys155 by Glu in the second EGF domain predicted by an A to G base exchange in the gene" Thrombosis Research. 70. 395-403 (1993)
• [Publications] Hidehiko Saito: "Human Ryudocan core protein:molecular cloning and characterization of the cDNA,and chromosomal localization of the gene" Biochemical and biophysical research communications. 190. 814-822 (1993)
• [Publications] Hidehiko Saito: "Genetic characterization of hereditary free protein S deficiency(abstract)" Blood. 82. 405a (1993)
• [Publications] M.Moroi: "Phorbol ester treatment of two megakaryoblastic cell lines,CMK and UT-7,induces specific protein composition changes with non-coincident time-courses" Biochem.Biophys.Acta. 1177. 139-146 (1993)
• [Publications] Hayashi,T.: "Protein S Tokushima:abnormal molecule with a substitution of Glu for Lys-155 in the second epidermal growth factor-like domain of protein S." Blood. 83. 683-690 (1994)
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• [Publications] Mimuro J: "An abnormal protein C(protein C Yonago)with amino acid substition of Gly for Arg-15 casused by a single base mutation of C to G in codon 57(CGG-GGG)" Int J Hematol. 57. 9-14 (1993)
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• [Publications] 藤村欣吾: "レセプター-基礎と臨床-" 井村裕夫,岡哲雄,芳賀達也,岸本英爾編, 944 (1993)
• [Publications] 藤村欣吾: "別刷・医学のあゆみ,血液疾患" 溝口秀昭,平井久丸,坂田洋一編, 462 (1993)
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• [Publications] 西村仁: "糖鎖の多様な世界" 木幡陽、箱守仙一郎、永井克孝 編, 257 (1993)
• [Publications] Yamamoto,S.: "Advances in Polyunsaturated Fatty Acid Research" Elsevier Science Publishers B.V., 294 (1993)
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• [Publications] Suzuki,K.: "Methods in Enzumology.(Protein C inhibitor.)" Academic Press,New York(eds.by Lorand.L and Mann.K.G.), 613 (1993)