Falk J, Townsend L E, Vogel L M, Boyer M, Olt S, Wease G L, Trevor K T, Seymour M, Glover J L, Bendick P J
Department of Surgery, William Beaumont Hospital, Royal Oak, Mich 48073, USA.
J Vasc Surg. 1998 May;27(5):902-8; discussion 908-9. doi: 10.1016/s0741-5214(98)70271-x.
A significant limitation to using genetically modified endothelial cells (ECs) to seed prosthetic grafts before implantation has been poor cell adherence to the graft lumen. Methodologic changes to improve cell adherence were evaluated in a canine carotid interposition graft model using 4 mm interior diameter expanded polytetrafluoroethylene.
ECs harvested from external jugular veins were grown in culture, with 80% of the cells from each culture transduced by incubation with an LXSN-type retroviral vector carrying a gene for human prourokinase and a neomycin resistance gene for selection in antibiotic G418. Control grafts had passive luminal coating with fibronectin and were seeded with transduced ECs immediately after G418 selection; these grafts were incubated for 2 days before implantation. Experimental grafts had fibronectin forcefully squeezed through the interstices and were seeded with ECs that had recovered in culture for 5 days after G418 selection; these grafts were incubated for 4 days before implantation. For each control (n = 9) and experimental (n = 12) graft, a graft prepared in the same fashion but seeded with the remaining autologous nontransduced cells was placed in the contralateral carotid artery. Grafts were explanted after 30 days and were evaluated for patency, thrombus-free surface area, and cell-free surface area.
No significant differences in patency rates were seen between any groups. The thrombus-free surface area was improved for experimental grafts (90%) compared with control grafts (76%), but this improvement did not achieve statistical significance. The cell-free surface area for transduced cells on experimental grafts was 65% compared with 96% for control grafts (p = 0.021) and was comparable with that for nontransduced cells on both control grafts (62%) and experimental grafts (51%; p = 0.201).
Adherence of genetically modified endothelial cells to small-diameter expanded polytetrafluoroethylene grafts in an in vivo physiologic flow model is significantly improved when cells have a more prolonged recovery from G418 selection, when the graft lumen is more uniformly coated with fibronectin before EC seeding, and when seeded grafts are left longer in culture before implantation to develop cell lining stability. The short-term patency rate of these seeded grafts is not affected by increased cell retention; long-term graft patency data and luminal healing require further evaluation.
在植入前使用基因改造的内皮细胞（ECs）接种人工血管时，一个显著的限制是细胞对血管腔的黏附性较差。在犬颈总动脉搭桥移植模型中，使用内径4毫米的膨体聚四氟乙烯评估了改善细胞黏附性的方法学变化。
从颈外静脉采集的内皮细胞在培养中生长，每种培养物中80%的细胞通过与携带人尿激酶原基因和新霉素抗性基因的LXSN型逆转录病毒载体孵育进行转导，新霉素抗性基因用于在抗生素G418中进行筛选。对照血管用纤连蛋白进行被动腔内包被，并在G418筛选后立即接种转导的内皮细胞；这些血管在植入前孵育2天。实验血管将纤连蛋白强力挤过间隙，并接种在G418筛选后在培养中恢复5天的内皮细胞；这些血管在植入前孵育4天。对于每个对照（n = 9）和实验（n = 12）血管，以相同方式制备但接种剩余自体未转导细胞的血管被置于对侧颈动脉。30天后取出血管，评估通畅率、无血栓表面积和无细胞表面积。
任何组之间的通畅率均无显著差异。与对照血管（76%）相比，实验血管的无血栓表面积有所改善（90%），但这种改善未达到统计学意义。实验血管上转导细胞的无细胞表面积为65%，而对照血管为96%（p = 0.021），并且与对照血管（62%）和实验血管（51%；p = 0.201）上未转导细胞的无细胞表面积相当。
在体内生理血流模型中，当细胞从G418筛选中恢复的时间更长、在接种内皮细胞之前血管腔用纤连蛋白更均匀地包被以及接种后的血管在植入前在培养中放置更长时间以形成细胞内衬稳定性时，基因改造的内皮细胞对小直径膨体聚四氟乙烯血管的黏附性显著改善。这些接种血管的短期通畅率不受细胞保留增加的影响；长期血管通畅数据和腔内愈合需要进一步评估。
