2000 Fiscal Year Annual Research Report

生体内分離冷却潅流法による肝臓特異的遺伝子導入-肝臓外科手技による遺伝子治療の可能性

Research Project

Project/Area Number	09307027
Research Institution	University of Ryukyus
Principal Investigator	白石祐之琉球大学, 医学部・附属病院, 講師 (00264482)
Co-Investigator(Kenkyū-buntansha)	廣安俊吾琉球大学, 医学部, 助手 (90295337) 武藤良弘琉球大学, 医学部, 教授 (60157724)
Keywords	アデノウイルスベクター / 遺伝子導入 / ブタ / ラット / von Willebrand factor / HVJ-リポソーム
Research Abstract	本研究での第1の目的として、これまでほとんど報告のない大動物の生体内肝臓に対する遺伝子導入を可能にすること。第2の目的として代謝性疾患の遺伝子治療を念頭に置き、肝臓に導入された遺伝子により目的蛋白を産生させ、これを代謝性疾患治療モデルとすることであった。これらの目的を達成するためには大動物疾患モデルが存在するvon Willebrand disease(vWD)の肝臓に欠損するvon Willebrand factor(vWF)遺伝子を導入しvWDブタの凝固異常を是正することを具体的な目的とした。vWFは血管内皮より産生・分泌され、肝内においては類洞内皮細胞に遺伝子導入される必要がある。マーカー遺伝子を運ぶadenovirus vector(AdV)もHVJ-liposome vector(HVJ)も、培養条件下のラット及びブタの血管内皮細胞に対して効果的な遺伝子導入が可能であった。ブタにおける遺伝子導入ではラットやマウスの小動物の報告と異なり、いずれのベクターも生体内で全身静注、門脈内投与などの血流中への一回投与方法では導入遺伝子(RT-PCR法)や発現蛋白(X-gal staining)を検出することはできなかった。われわれが検討した生体内分離肝灌流法は、バイオポンプを用いて門脈血・下大静脈血を体循環に灌流し、肝臓を体循環から分離し無血下に冷保存状態にするものである。この間に肝臓を高濃度のウイルスベクター(AdV;2x1011 pfu)と長時接触感染させ遺伝子導入効率を高めることができた。また肝内で肝細胞と肝類洞内皮の両者で蛋白発現を確認しえた。AdVに比べて遺伝子導入効率の低いRetro Virus Vectorにおいても生体内分離肝灌流法を用いることにより、ラット部分切除後の再生肝に対し高率の遺伝子導入効果を達成できた。また、同様の手技でブタの肝臓にヒトの補体抑制蛋白を導入し、これをヒト新鮮血で灌流する異種間肝移植の超急性拒絶モデルで、ヒト補体抑制蛋白の発現によるヒト補体抑制効果さらには肝内微小循環の改善効果を認めた。次に、ヒトvWFを運ぶHVJ-リポゾームベクター(アニオニック、カチオニック)を雑種ブタに投与した。同様に門脈内投与、肝動脈内投与、肝動脈内投与などの一回投与では、蛋白もmRNA(RT-PCR法)も検出できなかったが、生体内分離肝灌流法によりvWFのRNAが検出された。また、vWFをほぼ完全に欠損するvWDブタにおいても肝臓のみでなく肺や腎臓に対しても、一時的血流停止下に遺伝子導入を試みたが、遺伝子導入の証拠は得られなかった。

Research Products
(24 results)

All Other

All Publications (24 results)

[Publications] Shiraishi M: "Adenovirus-mediated gene transfer using in-situ perfusion of the liver graft"Transplant International. 10・3. 202-206 (1997)
[Publications] Shiraishi M: "Graft-directed gene transfection in a model of liver transplantation"Ryukyu Med J. 17・1. 21-24 (1997)
[Publications] Shiraishi M: "Successful Gene Transfer to the Porcine Liver in Vivo with an Adenoviral Vector"Journal of Surgical Research. 76・2. 105-110 (1998)
[Publications] Obuchi Y: "Highly Effective Adenoviral Mediated Gene Transfer to Procine Endothelial Cells In Vitro"Transplantation Proc. 30・7. 2917-2918 (1998)
[Publications] Nagahama M: "Adenoviral Mediated Gene Transfer to the Procine Liver in Vivo Using In Situ Perfusion of the Liver"Transplantation Proc. 30・7. 2914-2916 (1998)
[Publications] Taira K: "A New, Stable Model of Left Lobectomy for Living-Related Liver Transplantation in the Pig"Transplantation Proc. 30・7. 3207-3208 (1998)
[Publications] Nozato E: "In-situ perfusion of the liver under portosystemic shunt in rats : a stable model of cold ischemia for orthotopic liver transplantation"Transplantation Proc. 30・7. 3718-3720 (1998)
[Publications] Shiraishi M: "In-situ perfusion of the liver as a stable model of cold ischemia in orthotopic liver transplantation"Ryukyu Med J. 18・3. 73-77 (1998)
[Publications] Shiraishi M: "Early Detection of the Fas-FasL System in Hepatic Graft-Versus Host Disease After Rat Small-Bowel Transplantation"Transplantation Proc. 31・1-2. 567-568 (1999)
[Publications] Shiraishi M: "Stable Gene Expression with VSV-G Pseudotyped-Retrovirus Vector in the Rat Liver"Journal of Surgical Research. 84・2. 168-173 (1999)
[Publications] Hiroyasu S: "Analysis of the Fas System and Bcl-2 in Rat Liver Allograft Rejection"Journal of Surgical Research. 84・2. 204-211 (1999)
[Publications] 白石祐之: "ブタ肝臓異種灌流モデルにおける複数ヒト補体抑制蛋白遺伝子導入"外科治療. 81・3. 366-367 (1999)
[Publications] Shiraishi M: "Early Detection of Apoptosis and Fas Ligand Expression in Hepatic Graft-versus-Host Disease after Rat Small Bowel Transplantation"Journal of Surgical Research. 86・1. 136-144 (1999)
[Publications] Hiroyasu S: "Analysis of FAS System in Pulmonary Injury of Graft-Versus-Host Disease after Rat Intestinal Transplantation"Transplantation. 68・7. 933-938 (1999)
[Publications] Okuhama Y: "Protective effects of ulinastatin against ischemia-reperfusion injury"Journal of Surgical Research. 82・1. 34-42 (1999)
[Publications] Tomori H: "Protective effects lidocaine in hepatic ischemia/reperfusion injury in-vivo and ex-vivo"Ryukyu Med J. 19・1. 11-16 (1999)
[Publications] Tomori H: "In situ perfusion of the liver Enhances the Efficiency of retrovirus-mediated gene transfer to hepatocytes"Eur Surg Res. 32・1. 18-22 (1999)
[Publications] Higa T: "Limitations of Exogenous L-Arginie in Exerting a Cytoprotective Effect on Hepatic Ischemia/Reperfusion Injury"Surg Today. 30・4. 352-359 (2000)
[Publications] Mamadi T: "Effect of Bcl-2 expression on morphological alteration of hepatic mitochondria in liver regeneration after a partial hepatectomy"Ryukyu Med J. 19・4. 203-209 (2000)
[Publications] Nagahama M: "Adenovirus-mediated gene transfer of double human complement regulating proteins・DAF,CD59) in xenogeneic rat kidney perfusion"Transplant Proc. 32・5. 909-910 (2000)
[Publications] Shiraishi M: "Adenovirus-mediated gene transfer of the triple human complement regulating proteins・DAF,CD59,MCP)in xenogeneic pig liver perfusion"Transplant Proc. 32・5. 1116-1117 (2000)
[Publications] Shiraishi M: "Adenovirus-mediated gene transfer of Bcl-2 in graft-versus-host disease after rat intestinal transplantation"Transplant Proc. 32・6. 1263-1264 (2000)
[Publications] Shiraishi M: "Adenovirus-mediated gene transfer of DAF and CD59 in xenogeneic pig liver perfusion"Transplant Proc. 32・7. 2374-2375 (2000)
[Publications] Nagahama M: "Adenovirus-mediated triple gene transfer of human complement regulating proteins to the porcine endothelial cell"Transplant Proc. 32・7. 2511-2513 (2000)

2000 Fiscal Year Annual Research Report

生体内分離冷却潅流法による肝臓特異的遺伝子導入-肝臓外科手技による遺伝子治療の可能性

Principal Investigator

白石 祐之 琉球大学, 医学部・附属病院, 講師 (00264482)

Research Products

[Publications] Shiraishi M: "Adenovirus-mediated gene transfer using in-situ perfusion of the liver graft"Transplant International. 10・3. 202-206 (1997)

[Publications] Shiraishi M: "Graft-directed gene transfection in a model of liver transplantation"Ryukyu Med J. 17・1. 21-24 (1997)

[Publications] Shiraishi M: "Successful Gene Transfer to the Porcine Liver in Vivo with an Adenoviral Vector"Journal of Surgical Research. 76・2. 105-110 (1998)

[Publications] Obuchi Y: "Highly Effective Adenoviral Mediated Gene Transfer to Procine Endothelial Cells In Vitro"Transplantation Proc. 30・7. 2917-2918 (1998)

[Publications] Nagahama M: "Adenoviral Mediated Gene Transfer to the Procine Liver in Vivo Using In Situ Perfusion of the Liver"Transplantation Proc. 30・7. 2914-2916 (1998)

[Publications] Taira K: "A New, Stable Model of Left Lobectomy for Living-Related Liver Transplantation in the Pig"Transplantation Proc. 30・7. 3207-3208 (1998)

[Publications] Nozato E: "In-situ perfusion of the liver under portosystemic shunt in rats : a stable model of cold ischemia for orthotopic liver transplantation"Transplantation Proc. 30・7. 3718-3720 (1998)

[Publications] Shiraishi M: "In-situ perfusion of the liver as a stable model of cold ischemia in orthotopic liver transplantation"Ryukyu Med J. 18・3. 73-77 (1998)

[Publications] Shiraishi M: "Early Detection of the Fas-FasL System in Hepatic Graft-Versus Host Disease After Rat Small-Bowel Transplantation"Transplantation Proc. 31・1-2. 567-568 (1999)

[Publications] Shiraishi M: "Stable Gene Expression with VSV-G Pseudotyped-Retrovirus Vector in the Rat Liver"Journal of Surgical Research. 84・2. 168-173 (1999)

[Publications] Hiroyasu S: "Analysis of the Fas System and Bcl-2 in Rat Liver Allograft Rejection"Journal of Surgical Research. 84・2. 204-211 (1999)

[Publications] 白石祐之: "ブタ肝臓異種灌流モデルにおける複数ヒト補体抑制蛋白遺伝子導入"外科治療. 81・3. 366-367 (1999)

[Publications] Shiraishi M: "Early Detection of Apoptosis and Fas Ligand Expression in Hepatic Graft-versus-Host Disease after Rat Small Bowel Transplantation"Journal of Surgical Research. 86・1. 136-144 (1999)

[Publications] Hiroyasu S: "Analysis of FAS System in Pulmonary Injury of Graft-Versus-Host Disease after Rat Intestinal Transplantation"Transplantation. 68・7. 933-938 (1999)

[Publications] Okuhama Y: "Protective effects of ulinastatin against ischemia-reperfusion injury"Journal of Surgical Research. 82・1. 34-42 (1999)

[Publications] Tomori H: "Protective effects lidocaine in hepatic ischemia/reperfusion injury in-vivo and ex-vivo"Ryukyu Med J. 19・1. 11-16 (1999)

[Publications] Tomori H: "In situ perfusion of the liver Enhances the Efficiency of retrovirus-mediated gene transfer to hepatocytes"Eur Surg Res. 32・1. 18-22 (1999)

[Publications] Higa T: "Limitations of Exogenous L-Arginie in Exerting a Cytoprotective Effect on Hepatic Ischemia/Reperfusion Injury"Surg Today. 30・4. 352-359 (2000)

[Publications] Mamadi T: "Effect of Bcl-2 expression on morphological alteration of hepatic mitochondria in liver regeneration after a partial hepatectomy"Ryukyu Med J. 19・4. 203-209 (2000)

[Publications] Nagahama M: "Adenovirus-mediated gene transfer of double human complement regulating proteins・DAF,CD59) in xenogeneic rat kidney perfusion"Transplant Proc. 32・5. 909-910 (2000)

[Publications] Shiraishi M: "Adenovirus-mediated gene transfer of the triple human complement regulating proteins・DAF,CD59,MCP)in xenogeneic pig liver perfusion"Transplant Proc. 32・5. 1116-1117 (2000)

[Publications] Shiraishi M: "Adenovirus-mediated gene transfer of Bcl-2 in graft-versus-host disease after rat intestinal transplantation"Transplant Proc. 32・6. 1263-1264 (2000)

[Publications] Shiraishi M: "Adenovirus-mediated gene transfer of DAF and CD59 in xenogeneic pig liver perfusion"Transplant Proc. 32・7. 2374-2375 (2000)

[Publications] Nagahama M: "Adenovirus-mediated triple gene transfer of human complement regulating proteins to the porcine endothelial cell"Transplant Proc. 32・7. 2511-2513 (2000)

白石祐之琉球大学, 医学部・附属病院, 講師 (00264482)