tPA和VEGF165基因共表达质粒对人血管细胞增殖的影响和纤溶作用

目的：观察组织纤溶酶原激活物（tPA）和血管内皮细胞生长因子165（VEGF165）基因共表达质粒在血管平滑肌细胞（VSMC）中的表达，并研究表达产物对血管内皮细胞（VEC）和VSMC增殖的影响和纤溶作用。方法：用脂质体转染法将tPA和VEGF165的共表达质粒pBudCE4.1/tPA-VEGF165转染VSMC，逆转录－聚合酶链式反应（RT-PCR）和酶联免疫吸附实验（ELISA）分别从mRNA水平和蛋白质水平检测tPA和VEGF165的表达；纤溶蛋白板法检测转染基因的VSMC培养基中tPA的纤溶活性；取转染pBudCE4.1/tPA-VEGF165质粒的VSMC培养基培养VEC和VSMC，用四甲基偶氮唑盐（MTT法）和流式细胞技术检测转基因VSMC的细胞培养基对VEC和VSMC增殖的影响。结果：pBudCE4.1/tPA-VEGF165转染VSMC后，RT-PCR和ELISA检测发现，tPA和VEGF165在mRNA和蛋白质水平均有表达；转基因培养基有明显促进纤溶和促VEC增殖作用，而对VSMC增殖无作用。结论：tPA和VEGF165基因共表达质粒pBudCE4.1/tPA-VEGF165能在VSMC表达有生物学活性的tPA和VEGF165，为tPA和VEGF165基因转染防治移植心脏内血管狭窄奠定了基础。

Effects of co-expression plasmid of human tPA and VEGF165 on the proliferation of vascular cells and fibrinolysis activity

Objective To observe the expression of the co-expression plasmid of tissue-plasminogen activator (tPA) and vascular endothelia growth factor165 (VEGF165) in vascular smooth muscle cells (VSMC) and to study the effect of expressing products on the proliferation of VEC and VSMC and fibrinolysis activity. Methods The co-expression plasmid of tPA and VEGF165 (pBudCE4.1/tPA-VEGF165) was transfected into VSMC with the lipofection. The expression of tPA and VEGF165 at mRNA level was detected by RT-PCR and the protein level expression was detected by enzyme linked immunosorbent assay (ELISA). The fibrinolysis activity of culture medium of VSMC transfected tPA and VEGF165 genes was detected by fibrin plate technique. The VEC and VSMC were cultured with culture medium of VSMC transfected tPA and VEGF165 genes. And the proliferation of VEC and VSMC was evaluated with monoterazolium (MTT) and flow cytometry (FCM). Results The expression of tPA and VEGF165 at mRNA and protein levels in the transfected VSMC was demonstrated by RT-PCR and ELISA, respectively. The VSMC culture medium of transfected genes possessed evidently fibrinolysis activity. The expression products of tPA and VEGF165 in the VSMC had an evident effect on the VEC proliferation. But it had not an effect on the VSMC proliferation. Conclusion The eukaryotic co-expression plasmid of tPA and VEGF165 can be expressed in transfected VSMCs. The expression products have an obvious biological activity. The present study lay the foundation for future making use of tPA and VEGF165 to prevent and treat vascular stenosis in transplanted heart.