De Godoy J L, Malafosse R, Fabre M, Mitchell C, Mehtali M, Houssin D, Soubrane O
Laboratoire de Recherche Chirurgicale et de Biologie Hépatiques, CRI INSERM 94-05, Faculté de Médecine Cochin-Port Royal, Université Paris V, France.
Gene Ther. 2000 Nov;7(21):1816-23. doi: 10.1038/sj.gt.3301313.
Delivering retroviruses targeted to hepatocytes in vivo involves the injection of retroviruses directly into the portal vein. The aim of this work was to establish a clinically relevant system for retrovirus-mediated gene transfer in a new model of in vivo, in situ perfused rat liver and to study the transgene expression. At 24 h after partial hepatectomy, the liver was completely excluded from the splanchnic circulation using an extracorporeal shunt. Two independent normothermal, oxygenated perfusion systems were used. First, liver perfusion was carried out with a recirculating system (1 h). Culture supernatant containing retroviruses (1.5 x 10(8) ffu/ml, beta-galactosidase gene) was used as perfusate. Then the liver perfusion was maintained for more 30 min in a single liver passage system using culture medium without retroviruses as perfusate. High hepatocyte transduction rates (up to 34.4%) were obtained. PCR analysis showed no provirus in extrahepatic organs. Viral titrations performed simultaneously (inflow and outflow liver lines) showed that after 1 h of perfusion (up to 30 successive liver passages) retroviruses were still detected in the liver outflow perfusate (up to 2.0 x 10(7) ffu/ml). Washing the liver for 30 min dramatically decreased the leakage of retroviruses in the outflow. In order to be of clinical use, the injection of retroviruses targeted to hepatocytes in vivo should be done while the liver is completely excluded from the splanchnic circulation to avoid any extrahepatic retrovirus diffusion.
在体内将靶向肝细胞的逆转录病毒进行递送涉及将逆转录病毒直接注射到门静脉中。这项工作的目的是在一种新的体内原位灌注大鼠肝脏模型中建立一个与临床相关的逆转录病毒介导的基因转移系统，并研究转基因表达。在部分肝切除术后24小时，使用体外分流器将肝脏完全排除在内脏循环之外。使用了两个独立的常温、充氧灌注系统。首先，用循环系统进行肝脏灌注（1小时）。含有逆转录病毒（1.5×10⁸ffu/ml，β-半乳糖苷酶基因）的培养上清液用作灌注液。然后在单一肝脏通路系统中使用不含逆转录病毒的培养基作为灌注液，将肝脏灌注再维持30分钟以上。获得了较高的肝细胞转导率（高达34.4%）。PCR分析显示肝外器官中没有前病毒。同时进行的病毒滴定（流入和流出肝脏管路）表明，灌注1小时后（多达30次连续肝脏通路），在肝脏流出灌注液中仍可检测到逆转录病毒（高达2.0×10⁷ffu/ml）。用30分钟冲洗肝脏可显著减少逆转录病毒在流出液中的泄漏。为了用于临床，在体内将靶向肝细胞的逆转录病毒进行注射时，应在肝脏完全排除在内脏循环之外的情况下进行，以避免任何肝外逆转录病毒扩散。
