腺病毒介导uPA及VEGF165双基因转染提高脐静脉内皮纤溶活性的分子机制

The clinical efficacy of deep venous thrombosis(DVT) is poor, and seriously affecting the quality of life of patients. And the study of DVT is hot and difficult. The reason is that various thrombolytic agents can not fully reach the lesion and can not be effective to dissolve the clot and have low patency. Preliminary studies of our group indicated that transgenic uPA in vitro treatment significantly improved fibrinolysis, indicating that it can be applied to thrombolytic therapy. It has reported in the literature that VEGF165 inhibits intimal hyperplasia in vein, to promote endothelial repair and improve the patency rate. Therefore, we hypothesize that it would increase the efficacy and patency rate of venous thrombosis through thrombolysis by uPA and endothelial repair by VEGF165. The topic proposed to detect uPA and VEGF165 activity and cell signaling pathways by RT-PCR, Western blot and ELISA after adenovirus-mediated uPA and VEGF165 gene transfection in rat vena cava, and explore the molecular mechanisms of uPA and VEGF165 dissolve the clot and endothelial repair. It would lay the foundation to carry out gene therapy for clinical.

深静脉血栓形成（DVT）临床疗效差，严重影响患者生活质量,是目前国内外研究热点和难点。原因是各种溶栓剂不能充分到达病变部位，不能有效溶解血栓及通畅率低。我们的前期研究表明通过转基因uPA体外治疗可明显提高其纤溶活性，说明可应用于溶解血栓治疗。文献报道VEGF165可抑制静脉内膜增生，促进内皮修复，提高通畅率。因此，我们假设通过uPA的溶栓和VEGF165的内皮修复双重协同作用可望提高静脉内皮的纤溶活性，促进血栓的消融和提高通畅率。本研究将腺病毒介导的uPA和VEGF165双基因转染到大鼠下腔静脉内皮层，通过RT-PCR、Western Blot和ELISA等方法检测uPA和VEGF165的活性及细胞信号通路，探索uPA和VEGF165溶解血栓和内皮修复的分子机制，为临床开展基因治疗奠定基础。

uPA和VEGF165参与了溶解血栓和内皮修复，本课题通过细胞生物学方法和技术对uPA和VEGF165溶解血栓和内皮修复的分子机制进行了初步探索。首先构建uPA和VEGF165单基因真核表达质粒，并将uPA和VEGF165单基因真核表达质粒共转染至HUVECs，检测其转染后uPA、VEGF165的表达及对细胞增殖的影响。结果显示uPA 和 VEGF165单基因真核表达质粒共转染人脐静脉内皮细胞后目的基因uPA和VEGF165 有效表达，细胞增殖速率明显加强；在细胞内上调VEGF165能促进uPA的表达。推测VEGF165通过MAPK信号通路调uPA蛋白表达，进而应用Western Blot方法分析外源性VEGF165促进HUVECs细胞uPA蛋白表达的过程中MAPK 信号通路的作用。结果显示其中p38 MAPK在VEGF165促进HUVECs细胞uPA蛋白的表达具有重要作用。进一步构建基于Au@MSNs的溶栓药物控释系统，并在体内外评价其药物释放效率以及热溶栓的助溶栓作用。该药物控释系统在39°C环境下可以实现药物的快速释放，并且药物的释放量可达到90%，而在37°C环境下，该系统能保持良好的稳定性，体内外实验证实该体系在血栓处可高效的释放溶栓药物。综合上述结果，本研究为探讨uPA和VEGF165参与溶解血栓和内皮修复的机制奠定了坚实的工作基础。同时在溶栓药物的安全性输送方面取得更深入的认识，提供一种安全性好，溶栓效率高，具有潜在临床应用价值的血栓治疗新思路。项目资助发表SCI 2篇，待发表2篇。培养硕士生5名。项目投入经费48万元，支出9.89万元，剩余经费38.01万元，剩余经费计划用于本项目研究后续支出。