转基因血管内皮细胞的组织型纤溶酶原激活剂活性和种植黏附性研究  

Effects of mutant tPA gene transfer on tPA activity and adherence of endothelial cells seeded onto graft vessel

作　　者：陈力[1] 戴宁[2] 余红 陶思丰[1] 王建伟[1] 许斌[1] 

机构地区：[1]浙江大学医学院附属第二医院外科,杭州310009 [2]浙江大学医学院附属邵逸夫医院 [3]Department of Surgery

出　　处：《中华普通外科杂志》2003年第10期624-626,共3页Chinese Journal of General Surgery

基　　金：浙江省科研计划基金资助项目(991110 0 5 2 );浙江省医药卫生科学研究基金资助项目 (2 0 0 0A110 )

摘　　要：目的观察转基因血管内皮细胞 (endothelialcell,EC)的组织型纤溶酶原激活剂 (tissueplasminogenactivator ,tPA)活性和种植黏附性改变。 方法普通型和变异型tPA基因转染人大隐静脉EC ,ELISA法测定tPA活性。聚四氟乙烯人工血管腔面种植EC ,接入脉冲式体外灌注装置 1h ,比较EC保存率。结果普通EC组tPA活性为 (1 5± 1 0 )IU/ml,野生型和变异型tPA转基因组tPA活性分别为 (30 .0± 8.0 )IU/ml和 (14 1± 1 0 )IU/ml,转基因组均明显高于普通EC组 (P <0 0 1)。加入纤维蛋白后 ,变异型tPA组的tPA活性达 (5 0 .0± 19.0 )IU/ml,较加入纤维蛋白前(14 1± 1 0 )IU/ml明显提高 (P <0 0 1)。灌注 1h后 ,普通EC组EC保存率为 (45± 5 ) % ,野生型和变异型tPA组分别为 (39± 6 ) %和 (5 4± 13) % ,变异型tPA组高于普通EC组 (P <0 0 5 )。加入纤维蛋白后 ,变异型tPA组EC保存率下降至 (43± 8) % (P <0 0 1) ,但不低于野生型tPA组和普通EC组 (P >0 0 5 )。结论转基因内皮细胞tPA活性显著提高 ,变异型tPA的活性具特异性 ,有利于改善种植EC保存率。Objective To evaluate the activity of tissue plasminogen activator (tPA) gene transduced endothelial cells (EC) and the cell retention on graft vessel. MethodsKG1 EC were transduced with pseudotyped vectors carrying genes coding for either the wild type tPA or mutant tPA. The supernatants were collecteJPd and assayed for tPA activity in the presence and absence of fibrin. Polytetrafluoroethylene (PTFE) vessel grafts were seeded with EC, and then exposed to an in vitro flow system for 1 h. The number of EC on grafts were counted and the retention of EC were evaluated. ResultsWT5”BZ The tPA activity of the nontransduced EC was (1 5±1 0) IU/ml, while that of wild type tPA gene transduced EC increased to (30.0±8 0) IU/ml, mutant type tPA gene transduced EC increased to (14.1±1 0) IU/ml( P <0 001). In the presence of fibrin, however, the activity of zymogen mutant tPA reached to (50.0±19.0) IU/ml( P <0 01) that was higher than wild type tPA as well( P <0 05). After exposure to flow in the absence of fibrin, the cell retention rate was (45±5)% in nontransduced EC and (39±6)% in wild type tPA gene transduced EC ( P <0 05). Mutant zymogen tPA EC had an higher retention rate (54±13)% than the nontransduced EC ( P <0 05). When the cells were exposed to fibrin, the retention rate of mutant zymogen tPA EC was reduced to (43±8)% ( P <0 01). ConclusionWT5”BZ Intracellular transfer of the tPA gene can increase the tPA activity of EC. Zymogen tPA has specific protease activity against fibrin, and it can be used to increase thrombolytic activity of seeded cells and improve the retention of seeded EC on PTFE graft.

关 键 词：转基因 血管内皮细胞 组织型纤溶酶原激活剂活性 种植黏附性 人工血管 

分 类 号：R654.4[医药卫生—外科学]