转tPA基因血管内皮细胞的功能表达

The expression of tPA gene transduced into endothelium of ECV304

目的 观察组织型纤溶酶原激活剂（tPA）基因转导血管内皮细胞后的功能表达。方法 利用携带tPA基因的假型逆转录病毒转导血管内皮细胞株ECV304，用G418筛选法观察基因是否成功转导，采用发色底物显色法检测转基因ECV304细胞培养上清液中tPA纤溶活性变化。结果 转tPA基因后的血管内皮细胞经G418筛选后2周左右见大量克隆形成；转tPA基因ECV304细胞培养上清液中tPA纤溶活性在转导24h后增高，48h后增高更明显，72-96h浓度达高峰。结论 假型逆转录病毒载体成功介导tPA基因转导血管内皮细胞，并呈有效功能表达，为心血管手术后预防血栓形成的基因治疗提供实验依据。

Objective To investigate the expression of tissue-type plasminogen activator（tPA） gene transduced into vascular endothelium. Methods The cultured ECV304 was transduced with tPA gene using VSV-G/MuLV. The successful transduction was observed by means of G418 selection. The dynamic change of tPA activity of the supernatant of cultured cardiac fibroblasts after tPA gene transduction was measured with chromogenic peptide substrate assay. Results Clones of ECV304 transduced with tPA gene were formed about 2 weeks later after transduction. The tPA activity of the supernatant of gene-transduced ECV304 was increased 24 h later, and reached at the peak during 72-96 h after transduction. Conclusion tPA gene was successfully transduced into ECV304 using VSV-G/MuLV,and the expression of tPA gene in ECV304 was efficacious. It has laid good foundation for further research on the gene therapy for thrombosis after cardiovascular surgery.