| Project/Area Number |
09307027
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| Research Category |
Grant-in-Aid for Scientific Research (A).
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Digestive surgery
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| Research Institution | University of Ryukyus |
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Principal Investigator |
SHIRAISHI Masayuki (1999-2000) hospital attached to the college, University of Ryukyus, lecturer, 医学部・附属病院, 講師 (00264482)
草野 敏臣 (1997-1998) 琉球大学, 医学部, 助教授 (10244295)
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| Co-Investigator(Kenkyū-buntansha) |
HIROYASU Shunngo the college of medicine, University of Ryukyus assistant, 医学部, 助手 (90295337)
MUTO Yoshihiro the college of medicine, University of Ryukyus professor, 医学部, 教授 (60157724)
宮里 浩 琉球大学, 医学部, 助手 (90284976)
白石 祐之 琉球大学, 医学部・附属病院, 講師 (00264482)
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| Project Period (FY) |
1997 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥37,700,000 (Direct Cost: ¥37,700,000)
Fiscal Year 2000: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 1999: ¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 1998: ¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 1997: ¥23,300,000 (Direct Cost: ¥23,300,000)
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| Keywords | adenovirus vector / gene transfer / pig / rat / von Willebrand factor / HVJ-liposome / アデノウイルス / HVJリポソーム / レトロウイルス / in situ perfuison / adenoviral vector / retroviral vetor / gene transfer |
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| Research Abstract |
【Aims】 The aim of this study was to establish the effective gene transfer to a porcine liver in-vivo, and to utilize this model for gene therapy for metabolic disorder (a deficiency or abnormality of certain proteins or enzymes). In this setting, the transfected liver was expected to produce a target protein after successful gene transfer. To accomplish these aims, the von Willebrand factor (vWF) gene was transferred into the livers in-vivo, in pigs with and without von Willebrand disease (vWD), and the plasma vWF levels were thereafter assessed. Since vWF is produced and secreted from the endothelial cells, the vWF gene should thus be transferred into the sinusoidal endothelial cells in the pig liver.
【Results】 Preliminary studies have demonstrated that both the marker LacZ gene and human vWF were effectively transferred into the porcine endothelial cells in-vitro (≧ 90%), either with adenovirus vector (AdV) or HVJ-liposome vector (HVJ). On the other hand, neither the systemic injectio
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n nor the intraportal injection could establish any gene transfer (LacZ, vWF) in the in-vivo pig liver, using the same vectors (AdV or HVJ). We next tested in-situ perfusion of the liver (ISP) as a method for in-vivo gene transfer, in which the liver was isolated from systemic circulation under a pump driven bypass of the blood flow from the portal vein and inferior vena cava, and the liver was perfused with a high titer AdV or HVJ under bloodless and cold ischemic conditions. As a result, the marker LacZ expression was confirmed (X-gal staining ＆ RT-PCR) in both the hepatocytes and sinusoidal endothelial cells in the pig liver. The mRNA expression of transferred vWF (HVJ) was also confirmed by RT-PCR only after ISP.The plasma vWF levels, however, did not significantly increase after gene transfer.
【Conclusion】 The in-vivo gene transfer of vWF was thought to be possible by an ISP of the liver and HVJ-liposome vector. A significant increase of vWF, however, could not be confirmed, thus suggesting that further investigations are thus needed to establish the clinically relevant levels of gene transfer. Less
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