APPLICATION OF GENETRANSFER TECHNIQUE TO ENDOTHELIAL CELL SEEDED VASCULAR PROSTHESIS

用基因工程人内皮细胞（hECs）分泌抗血栓药物接种血管移植物具有相当大的临床潜力。用腺病毒载体将人血栓调节蛋白（hTM）和人组织型纤溶酶原激活剂（htPA）基因转染hEC，观察转染hEC的能力。用各种浓度的含有hTM或htPA基因的重组腺病毒感染平板上培养的hEC，以确定适当的转移条件。此外，将htPA基因转导的hEC接种到纤连蛋白涂覆的膨胀聚四氟乙烯移植物（内径4 mm）上，其中一些在体外暴露于脉动流以检查在回路中分泌htPA的能力，以及hEC在移植物上的保留。用30 pfu/ml的病毒汤可有效地将hTM和htPA基因转导到hEC中：hTM抗原589 ± 42.3 MGCC U/mg蛋白，hTM蛋白C激活辅因子活性32.4 ± 10.1 U/ml，htPA抗原4720 ± 86 ng/106 cells/6 h，htPA活性647 ± 32 IU/106 cells/6 h。转染htPA基因的内皮细胞在体外培养液中仍能分泌htPA。用tPA转导的EC灌注的移植物的保留率为84.0 ± 3.0%，用模拟感染（LacZ）的EC为94.2 ± 0.4%，用未感染的EC为82.1 ± 8.0%。这些保留值之间无显著差异。这些结果表明，用基因工程hEC接种小口径血管移植物具有增加体内局部抗血栓性质的潜力。

Seeding of vascular grafts with genetically engineered human endothelial cells (hECs) secretine anti-thrombotic agents has considerable clinical potential. Adenoviral vactor was used to transfer human thrombomodulin (hTM) or human tissue plasminogen activator (htPA) genes to hECs and the ability of the transduced hECs was examined. Cultured hECs on plates were infected with various concentrations of recombinant adenoviruses containing the hTM or htPA gene to determine the appropriate transfer conditions. Furthermore, htPA gene transduced hECs were seeded onto fibronectin-coated expanded polytetrafluoroethylene grafts (4mm internal diameter), some of which were exposed to pulsatile flow in vitro to check the ability of secreting htPA in the circuit, and retention of the hECs on the graft. Effective transduction of hTM and htPA gene into hECs was achieved with viral soup at 30 pfu/ml : hTM antigen 589+-42.3 MGCC U/mg protein, hTM protein C-activating cofactor activity 32.4+-10.1 U/ml, htPA antigen 4720+-86 ng/106 cells/6h, htPA activity 647+-32 IU/106 cells/6h. Grafts seeded with htPA gene transduced hECs retained the satisfactory secretion of htPA in the in vitro flow circuit. The retention of the perfused grafts with tPA transduced ECs was 84.0+-3.0%, with mock-infected (LacZ) ECs 94.2+-0.4%, and with the non-infected ECs 82.1+-8.0%. There were no significant differences among these retention values. There results indicate that seeding small-caliber vascular grafts with genetically engineered hECs has the potential to increase local anti-thrombotic properites in vivo.