Baer R P, Whitehill T E, Sarkar R, Sarkar M, Messina L M, Komorowski T A, Stanley J C
Department of Surgery, University of Michigan, Ann Arbor, MI, USA.
J Vasc Surg. 1996 Nov;24(5):892-9. doi: 10.1016/s0741-5214(96)70028-9.
An endothelialized lumen within a synthetic graft that expresses recombinant proteins with anticoagulant or antiproliferative activity has the potential to improve graft function. However, preliminary data suggest that genetic modification of endothelial cells (ECs) impairs their proliferation. The purpose of this investigation was to test the hypothesis that retroviral transduction of cultured ECs with the lac Z gene encoding for beta-galactosidase would decrease EC proliferation in vitro and graft endothelialization in vivo.
In vitro studies compared canine EC proliferation over a 14-day period among early-passage ECs (two and three) and late-passage ECs (six and nine) transduced with the BAG vector (containing the lac Z gene and the neomycin resistance gene), ECs transduced with the neomycin resistance gene only, the nontransduced ECs. In vivo canine studies assessed endothelialization of expanded polytetrafluoroethylene thoracoabdominal grafts seeded with autologous lac Z-transduced ECs (n = 7) or nontransduced ECs (n = 3) compared with that of nonseeded grafts (n = 3). Histochemical staining and DNA polymerase chain reaction was used 6 weeks after implantation to detect the presence of the lac Z gene in the grafts' cellular linings and perigraft tissues. Endothelialization was assessed by light microscopy and electron microscopy.
Proliferation of late-passage lac Z-transduced ECs in vitro was significantly decreased compared with neomycin resistance-transduced ECs or nontransduced ECs. Among early-passage ECs smaller but significant decreases in proliferation were noted among lac Z-transduced cells compared with nontransduced cells. Six of seven expanded polytetrafluoroethylene grafts seeded with transduced ECs showed lac Z gene expression. Lac Z gene expression was not found on grafts seeded with nontransduced ECs or nonseeded grafts. The endothelialized luminal surface area was significantly less in grafts seeded with lac Z-transduced ECs compared with grafts seeded with nontransduced ECs.
Retroviral-mediated transduction of canine ECs with the lac Z gene encoding for beta-galactosidase impairs EC proliferation in vitro and the ability of transduced ECs to form a confluent EC monolayer on the luminal surface of synthetic grafts in vivo.
合成移植物内具有抗凝或抗增殖活性重组蛋白表达能力的内皮化管腔，有改善移植物功能的潜力。然而，初步数据表明内皮细胞（ECs）的基因修饰会损害其增殖。本研究的目的是验证以下假设：用编码β-半乳糖苷酶的lac Z基因对培养的ECs进行逆转录病毒转导，会在体外降低ECs增殖，并在体内降低移植物的内皮化。
体外研究比较了用BAG载体（含lac Z基因和新霉素抗性基因）转导的早期传代ECs（第二代和第三代）和晚期传代ECs（第六代和第九代）、仅用新霉素抗性基因转导的ECs以及未转导的ECs在14天内犬ECs的增殖情况。体内犬类研究评估了植入自体lac Z转导ECs（n = 7）或未转导ECs（n = 3）的膨体聚四氟乙烯胸腹移植物的内皮化情况，并与未植入细胞的移植物（n = 3）进行比较。植入6周后，采用组织化学染色和DNA聚合酶链反应检测移植物细胞内衬和移植物周围组织中lac Z基因的存在情况。通过光学显微镜和电子显微镜评估内皮化情况。
与新霉素抗性转导的ECs或未转导的ECs相比，体外晚期传代lac Z转导的ECs增殖显著降低。在早期传代的ECs中，与未转导的细胞相比，lac Z转导的细胞增殖有较小但显著的降低。7个植入转导ECs的膨体聚四氟乙烯移植物中有6个显示出lac Z基因表达。在植入未转导ECs的移植物或未植入细胞的移植物中未发现lac Z基因表达。与植入未转导ECs的移植物相比，植入lac Z转导ECs的移植物内皮化管腔表面积显著更小。
用编码β-半乳糖苷酶的lac Z基因对犬ECs进行逆转录病毒介导的转导，会在体外损害ECs增殖，并在体内损害转导ECs在合成移植物管腔表面形成融合EC单层的能力。
