APPLICATION OF GENETRANSFER TECHNIQUE TO ENDOTHELIAL CELL SEEDED VASCULAR PROSTHESIS

用基因工程人内皮细胞（hec）分泌抗血栓药物播种血管移植物具有相当大的临床潜力。采用腺病毒载体将人血栓调节蛋白（hTM）或人组织纤溶酶原激活物（htPA）基因转染到hec细胞中，并检测转导的hec细胞的表达能力。用不同浓度的含hTM或htPA基因的重组腺病毒感染板上培养的hec，确定合适的转移条件。此外，将htPA基因转导的hec植入到纤维连接蛋白包被的膨化聚四氟乙烯移植物（内径4mm）上，其中一些移植物在体外暴露于脉冲流中，以检测电路中分泌htPA的能力，以及hec在移植物上的保留。用30 pfu/ml的病毒汤将hTM和htPA基因有效转导至hECs: hTM抗原589+-42.3 MGCC U/mg蛋白，hTM蛋白c活化辅助因子活性32.4+-10.1 U/ml， htPA抗原4720+-86 ng/106细胞/6h， htPA活性647+-32 IU/106细胞/6h。htPA基因转导的hec移植物在体外血流回路中保留了令人满意的htPA分泌。tPA转染的内皮细胞灌注移植物的保留率为84.0+-3.0%，模拟感染（LacZ）内皮细胞的保留率为94.2+-0.4%，未感染的内皮细胞的保留率为82.1+-8.0%。这些留存值之间没有显著差异。这些结果表明，用基因工程的hec播种小口径血管移植物有可能增加体内局部抗血栓特性。

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.