携tPA和VEGF165基因共表达质粒生物学功能研究

目的　观察组织纤溶酶原激活物(tPA)和血管内皮生长因子16 5 (VEGF16 5 )基因在血管内皮细胞(VEC)中的表达产物对VEC增殖和纤溶的影响。方法　将含有tPA和VEGF16 5的共表达质粒pBudCE4 1/tPA VEGF16 5导入VEC中,RT PCR法和Westernblot法分别从mRNA水平和蛋白质水平检测被转染VEC中的tPA和VEGF16 5表达;纤溶蛋白板法检测转基因VEC培养液的纤溶活性;用3 H胸腺嘧啶核苷掺入法( 3 H TdR)和流式细胞技术观察转基因VEC培养液对VEC和VSMC增殖的影响。结果　pBudCE4 1/tPA VEGF16 5转染VEC后,tPA和VEGF16 5分别在mRNA和蛋白质水平均有表达;转基因培养液有纤溶活性,并对VEC增殖有显著作用,而对VSMC增殖无作用。结论　pBudCE4 1/tPA VEGF16 5能在VEC表达出有生物学活性的tPA和VEGF16 5。

A study on biological activity of co-expression plasmid of human tissue plasminogen activator and vascular endothelial growth factor 165

Objective To observe the co-expression plasmid of tissue plasminogen activator (tPA) and vascular endothelia growth factor165 (VEGF165) in vascular endothelial cell (VEC) and to study the effect of the product on the proliferation of VEC and fibrinolysis activity. Methods pBudCE4.1/tPA-VEGF165 was transfected into VECs by using lipofection. The expression of tPA and VEGF165 at mRNA level was detected by RT-PCR and expression at protein level was detected by Western blot. The fibrinolysis activity of VEC culture solution of transfecting tPA and VEGF165 genes were detected by fibrin plate technique. The VEC and VSMC were cultured with VEC culture solution of transforming tPA and VEGF165 genes, the proliferation of VEC and VSMC were evaluated with 3?H-TdR incorporation and flow cytometry (FCM). Results The expression of tPA and VEGF165 in the transfected VECs was detected. The fibrinolysis activity of transfected VEC culture solution was also detected. tPA and VEGF165 products in VECs elevated proliferation of VEC, while there was no effect on the proliferation of VSMC. Conclusion The tPA and VEGF165 eukaryotic co-expression plasmid could express in transfected VECs, and the expression products have biology activity.