遺伝子制御による異種臓器移植の研究

1997 Fiscal Year Annual Research Report

• Project/Area Number: 08407030
• Research Institution: Nagoya University
• Principal Investigator: 高木 弘 名古屋大学, 医学部, 教授 (70154755)
• Co-Investigator(Kenkyū-buntansha): 岡田 秀親 名古屋市立大学, 医学部, 教授 (30160683) ; 横山 逸男 名古屋大学, 医学部, 講師 (60240206) ; 鬼頭 純三 名古屋大学, 医学部, 教授 (60022802) ; 村松 喬 名古屋大学, 医学部, 教授 (00030891) ; 中島 泉 名古屋大学, 医学部, 教授 (40022826)
• Keywords: 異種移植 / トランスジェニック動物 / 補体不活化因子 / 異種抗原 / ガラクトシルトランスフェラーゼ / フコシルトランスフェラーゼ

Research Abstract

移植医療におけるドナー不足は深刻な問題であり、世界中で動物の臓器を利用する異種移植研究が活発に行われている。ブタがドナーとして最適であると考えられているが、激烈なる免疫反応を制御することが最大の課題である。レシピエントに存在する抗ブタ自然抗体および補体の活性化による超急性拒絶反応、血管内皮細胞、凝固系の活性化、サイトカイン、細胞性反応による遅延型拒絶反応の問題を解決する必要がある。
遺伝子制御による異種移植の臨床応用に少しでも近づけるためにの有用な情報を提供することが本研究の目的である。
拒絶反応を抑制するためのヒト補体制御因子を導入したトランスジェニックブタは、既に作出に成功しており、異種抗原であるαガラクトース(αGal)抗原発現を抑制したブタの作出を試みている。私どもは、FT遺伝子トランスジェニックブタ作出、アデノウイルスを用いたFT遺伝子やGTリポザイムの導入、GT遺伝子の代わりにFT遺伝子を導入するノックインの手法を開発した。
αGal以外の抗ブタ抗体の可能性として、Hanganutzlu-Deicher(HD)抗原をブタの血管内皮細胞に証明した。ヒトにはこの抗原は存在せず、一部のヒトにおいてHD抗体の存在が明らかにされ、αGal抗原を除去した後に問題となりうることが示唆された。しかし、ヒヒにもHD抗原が存在することから、HD抗原抗体反応についてはヒヒはヒトのモデルとなりえないことが明らかにされた。
前臨床試験としての霊長類(ヒヒ)を用いた移植実験モデルを確立するために、ヒヒの血液検査、ウイルス検査を行った。生化学、補体活性はヒトとほぼ同じであり、抗ブタ抗体においても超急性拒絶反応に重要な役割を果たすIgMはヒトとほぼ同程度に保有しており(IgGはヒトと比較して低い)、ブタからヒトへの移植モデルになりうると考えられた。ウイルス抗体検査においても、一部Bウイルス抗体の陽性結果が出たが、HSVとの交差反応であると考えられ、実験の安全性についても問題を認めなかった。
遺伝子操作により、ドナーブタを移植に適するように形質転換する研究が行われているが、主要な抗ブタ抗体の結合するαガラクトース抗原の発現を完全に消去するブタは今の所完成していない。そのため、移植前に抗ブタ抗体の除去は必要不可欠である。ブタの臓器を用いた灌流では、腎臓で約30%、肝臓で約50%の抗体が除去された。ABO不適合移植の臨床で抗A、抗B抗体を除去するために使用されているDFPP(double filtration plasmapheresis)では95%以上の抗ブタ抗体除去効率が示され、その有用性が示された。
急性肝不全の治療としての補助的同所性肝移植モデルを確立し、タンパク分画によりブタ、ヒヒの肝細胞により合成されるタンパクを区別することに成功した。
異種移植拒絶反応を抑制するための基礎研究を多方面から推進し、今後遺伝子操作により拒絶反応の起しにくい形質転換ドナーブタから霊長類(ヒヒ)へのより臨床に近い形で研究を進める必要がある。

Research Products

• [Publications] 林 衆治, 高木 弘: "遺伝子制御による異種移植の研究" 日本消化器外科学会雑誌. 29-4. 907-910 (1996)
• [Publications] I Yokoyama, H Takagi: "Beneficlal effect of donor pretreatment with thromboxane A2 synthase inhibitor on the graft survival in pig liver transplantation." Journal of Surgical Research. 60. 232-238 (1996)
• [Publications] S Hayashi, H Takagi: "Inhibitory effcct of double transfection to xenoendothelial cells using both decay accelerating factor and homologous restriction factor 20 genes on complement dependent" Journal of Surgical Research. 61. 165-169 (1996)
• [Publications] I Yokoyama, H Takagi: "Liberation of vasoactivc substances and its prevention with thromboxane A2 synthase inhibitor in pig liver transplantation." Transplantation International. 9. 76-81 (1996)
• [Publications] 小林孝彰, 高木弘: "異種臓器移植におけるトランスジェニックブタ作製の重要性" 細胞工学. 15. 81-89 (1996)
• [Publications] Y Taminaga, H Takagi: "Molecuitar genetics of hyperparathyroid disease Current Opinion in Nephrology and Hypertention" Current Opin Nephrol Hypertiens. 5. 336-341 (1996)
• [Publications] S Hayashi, H Takagi: "Inhibition of human complement dependent cell lysis by bovine aortic endothelial cells transfected with membrane-bound complement regulatory factor (DAF and HRF20) gene using a retroviral vector" Enropean Surgical Research. 28. 440-446 (1996)
• [Publications] I Yokoyma, H.Takagi: "Decreased fas antigen expression of culutred hapatocytes with FK506" Transplantation Proceesings. 28-6. 3189-3190 (1996)
• [Publications] S Hiraga, H Takagi, S kitagawa: "Study of the status and Certification of transplant coordinators in Japan." Transplantation Proceeding. 28-1. 260-263 (1996)
• [Publications] M Negita, H Takagi: "Retrovirus-mediated human superoxide dismutase cDNA transfer to prevent ischemia-reperfusion injury." Transplantation Proceedings. 28-1. 342 (1996)
• [Publications] S Hayashi, H Takagi: "Application of a vena cava allograft in split liver transplantation in pigs." Transplantation Proceedings. 28-1. 330 (1996)
• [Publications] M Negita, H Takagi: "Measurement of the ratio of superoxide-scavenging activity in the graft in swine liver transplantation." Transplantation Proccedings. 28-1. 329 (1996)
• [Publications] C Koike, H Takagi: "Introduction of α(1,2)-fucosyltransferace and its effect on α-Gal epitopes in transgenic pig." Xenotransplantation. 3. 81-86 (1996)
• [Publications] I Yokoyama, H Takagi: "Factors affecting graft function in cadaveric renal transplantation from non-heart-beating donors using a double balloon catheter." Transplantation Proceedings. 28-1. 116-117 (1996)
• [Publications] S Hayashi, H Takagi: "Genetic engineering targeted to donor organs using adenoviral vector." Transplantation Proccedings. 28-1. 215 (1996)
• [Publications] S Hayashi, H Takagi: "Tissue distribution of Gal α(1,3) Gal epitope in heart,kidney,and liverk of pig and mouse." Transplantation Proccedings. 28-1. 216 (1996)
• [Publications] 横山逸男, 高木 弘: "死体肝移植とシクロスポリンの臨床" シクロスポリンの実際 国際医学出版. 225-269 (1996)
• [Publications] 横山逸男, 高木 弘: "免疫抑制剤と移植" 医系微生物学朝倉書店. 第二版. 159-161 (1996)
• [Publications] 高木 弘: "シクロスポリンの登場と移植の発展:シクロスポリンの実際" 国際医学出版. 3-13 (1996)
• [Publications] C Koike, H Takagi: "Establishment of a human DAF/HRF20 double transgenic mouse line is not sufficient to suppress hyperacute rejection" Surgery Today. 26. 993-998 (1996)
• [Publications] Y Namii, H Takagi: "Tetrahydropyranyladriamycin with FK 506 combination therapy for hamster-to-rat concordant xenotransplantation" Transplantation Proceedings. 28-2. 709-710 (1996)
• [Publications] C Koike, H Takagi: "How can human DAF and HRF20 prevent HAR in transgenic mice?" Transplantation Proceedings. 28-2. 509-600 (1996)
• [Publications] C Koike, H Takagi: "Converting α-Gal epitope of pig into H antigen" Transplantation Proceedings. 28-2. 553 (1996)
• [Publications] S Hayashi, H Takagi: "Effect of antisense ribozyme to pig α(1,3) galactossyl transferase gene on the expression of Gal α(1,3)Gal epitope" Transplantation Proceedings. 28-2. 628 (1996)
• [Publications] 高木 弘: "異種移植" 医学のあゆみ. 177-3. 258-259 (1996)
• [Publications] 冨永芳博, 高木 弘: "腎性上皮小体機能亢進症の外科的治療" 腎と透析. 40. 92-96 (1996)
• [Publications] 福宜田正隆, 高木 弘: "肝移植における臓器保存の方向と将来" 国際医書出版. 45-58 (1996)
• [Publications] I Yokoyama, H Takagi: "Hyperamylasemia in kidney transplant recipients." Transplantation Proceedings. 28-3. 1476-147 (1996)
• [Publications] S Hayashi, H Takagi: "Possibility of gene transfer to the xenogeneic liver of guinea pig and pig using adenoviral vector." Transplantation Proceedings. 28-3. 1440 (1996)
• [Publications] K Uchida, H Takagi: "OKT3 rescue therapy for 63 rafractory rejections in 405 renal allografts" Transplantation Proceedings. 28-3. 1358-1359 (1996)
• [Publications] S Hayashi, H Takagi: "Split liver transplantation in pigs with reconstruction of the inferior vena cava" Transplantation Proceedings. 28-3. 1798 (1996)
• [Publications] I Yokoyama, H Takagi: "Fas antigen expression and apoptosis inductioc of in vitro cultured hepatocytes with high concentrations of cycrosporine A" Transplantation Proceedings. 28-3. 1383-138 (1996)
• [Publications] 高木 弘: "免疫抑制剤シクロスポリン" 今日の移植. 9-4. 431-432 (1996)
• [Publications] H Takagi: "XENO-Xenotransplantation reserch in Japan" XENO. 4-4. 62-63 (1996)
• [Publications] 小林孝彰, 高木 弘, 浅野浩史: "免疫抑制剤投与時における真菌症" 臨床と微生物. 23-5. 511-516 (1996)
• [Publications] T Kobayashi, Takagi H: "TAP1,TAP2,LMP2,DMA,and DMB Genetic Polymorphisms in Renal Transplantation" Transplantation Proceedings. 30. 29-30 (1996)
• [Publications] Y Tominaga, Takagi H: "Clonal Analysis of Nodular Parathyroid Hyperplasia in Renal Hyperparathroidism" World journal of Surgery. 20. 744-752 (1996)
• [Publications] T Kojima, Takagi H: "Role of Fc Fragments in Inhibition of Xenogenic Hypeeracute Rejection by Intravenous Imunoglobulin" Transplantation. 62-5. 559-562 (1996)
• [Publications] S Taniguchi, Takagi H: "In vivo Immunoadsorption of Antipig Antibodies in Baboons Using a Specofic GaL-α 1-3Gal Column" Transplantation. 62-10. 1379-138 (1996)
• [Publications] Yokoyama I, Takagi H: "Apoptosis in pig endothelial cells after reperfusion and its prevention by genetic induction of human Cu,Zn-SOD cDNA." Transplantation Proceedings. 29. 1364 (1997)
• [Publications] Morozumi K, Takagi H, Uchida K: "Electron-microscopic peritubular capillary lesion in a specific and uself diagnostic indicator for chronic rejection of renal allografts showing less specific morphologic lesion in the cyclosporin era." Transplantation Proceedings. 29. 1389-92 (1997)
• [Publications] Takeda A, Takagi H: "Sevare cyclosporine arteriolopathy with focal segmental glomerulosclerosis is not a fatal finding in chronic renal allograft failure after year 5 of transplantation using cyciosporine." Transplantation Proceedings. 29. 96-99 (1997)
• [Publications] Hayashi S, Takagi H: "Effect of antisense ribozyme to alpha (1,3) galactosyl transferase gene on the expression of GAl alpha (1,3) GAL epitope." Transplantation Proceedings. 29. 893 (1997)
• [Publications] Koike C, Takagi H: "Reduction of α-gal epitopes in transgenic pig by introduction of fuman α 1-2 fucosyltransterase." Transplantation Proceedings. 29. 894 (1997)
• [Publications] Negita M, Takagi H: "Protective effect of human suoeroxide dismutase cDNA transfection in the prevention of cold preservation injury." Transplantation Proceedings. 29. 1363 (1997)
• [Publications] T Kojima, H Takagi: "Inhibition of completment-mediated immune hemolysis by the constant domain of immunoglobuin" Peptido Chemistry. (1997)
• [Publications] T Nagasaka, H Takagi: "α-garactosyl oligosaccharides conjugated with polyethylene glycol as potential inhibitors of hyperacute upon xenotransplantation" BIOMEDICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. 232. 731-736 (1997)
• [Publications] 小林孝彰, 高木 弘: "HLAのマッチングと臓器移植検査と技術-増刊号-輸血検査実践" マニュアル医学書院. 25-7. 120-121 (1997)
• [Publications] S Hayashi, H Takagi: "Adenovirus-mediated gene transfer to the xenogeneic liver in liver xenotransplantation : The transductio of complement regulatory factor genes (DAF and HRF20)" Transplantation Proceeding. 29. 2211 (1997)
• [Publications] S Hayashi, H Takagi: "Effect of CTLA4-Ig gene transfer using adenoviral vector in organ and cell transplantation" Transplantation Proceeding. 29. 2212 (1997)
• [Publications] S Hayashi, H Takagi: "Adenovirus-mediated gene transfer of antisense ribozyme for α(1,3) garactosyl transferase gene and (1,2) fucosyltransferase gene in xenotranplantation" Transplantation Proceeding. 29. 2213 (1997)
• [Publications] Y Namii, H Takagi: "Evidence that the adenoviral vector containing the CTLA-Ig gene improves transgene expression and graft survival" Transplantation Proceeding. 29. 1738-1739 (1997)
• [Publications] 林 衆治、高木 弘: "特集 寛容導入をめざす移植免疫学-ヒト遺伝子導入による異種拒絶反応制御" Molecular Medicine. 34-7. (1997)
• [Publications] Katayama A, Takagi H: "tudies of α-Gal Dependent Hyperacute Xenotransplant Rejection Using DOuble Knockout Embryonic Stem Cells" Transplantation Proceeding. 29. 2262 (1997)
• [Publications] Chihiro Koike, Hiroshi Takagi: "Direct gene replacement of the mouse α(1,3)-galactsyltransgferase gene with human α(1,2)-fucosyltransgferase gene : Converting α galactsyl epitopes into H antigens" Xenotransplantation. 4. 147-153 (1997)
• [Publications] H Asano, H Takagi: "Significance of tumor necrosis factor microsatellite polymorphism in renal transplantation" TISSUE ANTIGENS. 50. 484-488 (1997)
• [Publications] Shuji Hayashi, Hiroshi Takagi: "Inhibition of establishment of Hepatic metastasis in mice by combination gene therapy using both herpes simplex virusthymidine kinase and granulocyte macrophage-colony stimulating factor gene in mune colon cancer" Cancer Genen Therapy. 4-6. 339-344 (1997)
• [Publications] 小林孝彰、高木 弘: "ABO血液型不適合腎移植と異種移植の接点" 今日の移植. 10-6. 947-951 (1997)
• [Publications] M Negita, Takagi H: "Protective Effect of Human Superoxide Dismutase cDNA Transfection in the Prevention of Cold Preservation Injury" Transplantation Proceedings. 29. 1363 (1997)
• [Publications] Takagi H: "Organ Transplants Still Too in Japan and Asian Countries" Transplantation Proceedings. 29. 1580-158 (1997)
• [Publications] I Yokoyama, Takagi H: "Measurement of portal venous flow velocity with an implantable miniature Doppler probe in pig liver transplantation" Transplantation international. 10. 116-120 (1997)
• [Publications] Takagi H: "Evalution of Tetrahydropyranyladriamycin as an Immunosuppressant in Concordant Xenotransplantation" World journal of Surgery. 16. 283-289 (1997)
• [Publications] S Hayashi, Takagi H: "Effect of Gene with the Herpes Simplex Virus-Thymidine Kinase Gene on Hepatic Metastasis in Murine Colon Cancer" Surgery Today. 27. 40-43 (1997)
• [Publications] K Uchida, Takagi H: "Renal Transplantatio and Secondary Hyperparathyroidism" Seminars in Surgical Oncology. 13. 97-103 (1997)
• [Publications] Y Tominaga, Takagi H: "Histopathology,Pathophysiology,and Indications for Surgical Treatment of Renal Hyperparathyroidism" Seminars in Surgical Oncology. 13. 78-86 (1997)
• [Publications] Takagi H: "PARATHYROID HYPERPLSIA AND TUMOR" Seminars in Surgical Oncology. 13. 71-72 (1997)
• [Publications] Y Tominaga, Takagi H: "Surgical Treatment of Renal Hyperparathroidism" Seminars in Surgical Oncology. 13. 87-96 (1997)
• [Publications] Y Tachi, Takagi H: "Variability of Cytotoxicity to Pig Cultured Cells and Its Determinant Factor in Human Sera" Transplantation Proceedings. 30. 71-73 (1998)
• [Publications] Yokoyama I, Takagi H: "Transplant Portal venous flow velocity measured by the implantable miniature Doppler probe in pig liver transplantation." International. (in press).
• [Publications] Yokoyama I, Takagi H: "Prevention of endothelial cell apoptosis with genetic induction of human Cu,Zn-SOD cDNA." Transplantation. (in press).
• [Publications] Yokoyama I, Takagi H: "Portal venous flow velocity measured by the implantable miniature Doppler probe in pig liver transplantation." International. (in press).
• [Publications] Yokoyama I, Takagi H: "Fas antigen expression and its modification by immunosuppressants." Dig Dis Sci. (in press).
• [Publications] S Hayashi, H Takagi: "Protection of guinea pig cell from rat complement-mediated lysis by the transfection of rat complement regulatory factor512 gene Xenotransplantation" Xenotransplantation. 3. 217-221 (1996)
• [Publications] 高木 弘: "異種移植への展望" 臨床透析. 12-3. 269-270 (1996)
• [Publications] 横山逸男, 高木 弘: "異種移植" 日本外科学会雑誌. 97-11. 1010-101 (1996)
• [Publications] M Negita, H Takagi: "Human superoxide dismutase cDNA transfection and its in vitro effect on cold preservation." Biomedical and biophysical resarch communication. 218. 653-657 (1996)
• [Publications] Kobayashi, Takagi H, and Cooper DKC: "Investigation of the anti-complement agents,FUT-175 and K76COOH,in discordant xenotransplantation" Xenotransplantation. 3. 237-245 (1996)
• [Publications] Hayashi S, Takagi H: "Protection of guinea pig cells transfected with the 512 gene for complement dependent cytolysis in rats." Transplantation Proceedings. 28. 592 (1996)
• [Publications] Morozumi k, Takagi H: "A comparative study of morphological characteristics of renal injuries of tacrolimus (FK506) and cyclosporin (CyA) in renal and CyA nephrotoxicity similar?" Transplantation Proceedings. 28. 1076-1078 (1996)
• [Publications] Yokoyama I, Takagi H: "Liver transplantation and hepatocellular carcinoma." Sem Surg Oncol. 12. 212-216 (1996)
• [Publications] Yokoyama I, Takagi H: "Decresed Fas antigen expression of the cultured hepatocytes with tacrolimus (FK506)" Transplantation Proceedings. 28. 3191-3193 (1996)
• [Publications] 横山逸男、高木 弘: "肝細胞癌に対する肝移植" 肝胆膵. 33. 55-61 (1996)
• [Publications] S Hayashi, H Takagi: "Genetic Enginnering targeted to donor organs using" Transplantation Proceeding. 28-28. 225- (1996)
• [Publications] T Kobayashi, H Takagi, D.K.C.Cooper: "In vitro and vivo investigation of anticomplement agents FUT-175 and K76COOH,in the prevention of hyperacute rejection following discordant xenotransplantation in a nonhuman primate model" Transplantation Proceeding. 28-2. 604 (1996)