逆转录病毒载体组织型纤溶酶原激活剂基因的局部表达及溶栓效果

Local Epression and Thrombolysis of Retroviral Vector with Tissue Type Plasminogen Activator Gene

目的:研究逆转录病毒载体组织型纤溶酶原激活剂基因的局部表达及溶栓效果。方法:70只大耳白兔随机分为A、B、C 3组,分别为30、20、20只。局部基因转导后,分别于术后2d和75d每组中半数动物采集血液、下腔静脉冰冻切片聚光共聚焦观察增强型绿色荧光蛋白(EGFP)表达、Dacron片于光镜及电镜下观察表面血栓情况、Western blot及ELISA法检测外源组织型纤溶酶原激活剂(tPA)表达、tPA活性及含量测定。结果:术后2d和75d取材,EGFP表达:A组多而强,B组也有,C组无。血栓:A组Dacron片表面均无血栓,B组表面均有血栓。tPA表达:A组均有外源表达,B组未检测到。血浆平板显示:A组均有大的溶解圈,有明显溶栓作用,B组没有;根据蚓激酶计算出的纤溶活性显示:A组术后2d和75d所取静脉tPA活性两时间点比较,差异无统计学意义(P>0.05)。术后2d和75d A组、两对照组中6个亚组所取静脉tPA含量治疗组明显高于对照组(P<0.01);A组中两时间点比较,tPA含量差异无统计学意义(P>0.05)。结论:pLEGFP-N1-tPA局部基因转导能有效阻止外源移植物表面血栓形成。

Objective：To study the local expression and thrombolysis of retroviral vector with tissue type plasminogen activator gene. Methods：70 New Zealand white rabbits were randomly divided into 3 groups .Group A included 30 rabbits, group B 20 and group C 20. Samples of blood, Dacron pieces and inferior caval veins from half of above in each group were harvested on second day and another half on 75th day after local gene transferred. The EGFP expression of harvested inferior caval veins were observed under the laser microscopy. The thromuses on the surfaces of Dacron patches were detected by stereoscope and electron microscope. The tPA expression in inferior caval veins was detected by Western blot and their thrombolysis and activities were observed and calculated in plasma plates. ELISA was used to determine the contents of tPA. Results：At the second and 75th day, much and strong EGFPs in inferior eaval veins of group A were observed and so did in group B. No EGFPs were seen in group C. 70 Dacron patches were magnified.There were no thrombuses on the surfaces of Dacron patches in group A but there were many thromhuses in group B. The expression of tPA was found that exogenous tPA highly expressed in group A but not in group B. According to the plasma plates, big areas of thrombotysis were found in group A, indicating strong thrombolysis. On the contrary, there was no thrombolysis in group B. According to the activity of lumbrukinase, there was no significant difference in activities oftPA between 2rid and 75th days （P〉0.05）. The tPA contents in inferior caval veins from 6 sub-units were obviously higher than in control group （P〈0.01）. There was no significant difference in tPA contents between 2nd and 75th days in group A （P〉0.05）. Conclusions：pLEGFP-Nl-tPA local transferring can effectively prevent thrombosis on the surface of exogenous transplant.