组织型纤溶酶原激活剂基因逆转录病毒载体体内靶向溶栓  

Target thrombolysis of retroviral vector with tissue type plasminogen activator gene on local thrombus

作　　者：张凯伦[1] 龚永生[1] 蒋雄刚[1] 刘小斌[2] 厉泉[1] 徐磊[1] 张晓明[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院心血管外科,武汉430022 [2]湖北省十堰市人民医院胸心外科

出　　处：《中华实验外科杂志》2008年第9期1091-1094,F0003,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30571838）

摘　　要：目的观察组织型纤溶酶原激活剂（tPA）基因局部转导对特氟隆（Dacron）片（与机械瓣瓣环同质）的溶血栓作用。方法70只兔建立下腔静脉内Dacron片植入血栓模型，随机分为pLEGFP-N1-tPA治疗组（n=30）、pLEGFP-N1空载体对照组（n=20）、空白对照组（n=20），局部基因转导，于术后2、75d各组一半动物取材（6个亚组A1、B1、C1、A2、B2、C2），聚光共聚焦（confocal）观察所取静脉增强型绿色荧光蛋白（EGFP）表达，体视镜和电镜观察Dacron片表面血栓情况，免疫印迹（Western blot）、血浆平板和酶联免疫吸附实验（ELISA）分别检测所取静脉tPA表达、溶栓、活性及含量变化。结果术后2d和75d取材，治疗组所取静脉，confocal均观察到多而强的EGFP表达，pLEGFP-N1对照组也有EGFP表达，但空白对照组无EGFP表达。70个Dacron片加未植入组10个Dacron片共80个，在体视镜（×160倍）和电镜（×500倍）下观察，未植入组和治疗组Dacron片表面均无血栓，对照组表面均有血栓。治疗组所取静脉Western blot检测均有外源tPA表达，对照组未检测到。血浆平板显示：治疗组均有大的溶解圈，有明显溶栓作用，对照组没有；根据蚓激酶计算出的纤溶活性显示：治疗组术后2d和75d所取静脉tPA活性分别为（529．62±9．05）、（537．50±12．45）U／g，两时间点比较，差异无统计学意义（P〉0．05）。术后2d和75d治疗组、两对照组中6个亚组所取静脉tPA含量分别为（A1 737．64±13．19）、（B1 29．88±5．61）、（C1 28．71±5．49）、（A2 742．87±10．56）、（B2 32．03±6．26）、（C2 31．34±5．63）ng／g，治疗组明显高于对照组（P〈0．01）；治疗组中两时间点比较，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 identify the target thrombolysis of recombinant tissue type plasminogen activator （tPA） transferred into the tissue around the Dacron patch （the same materials made of the ring of mechanical valve） in inferior caval vein in vivo. Methods Seventy Dacron patches were transplanted into the inferior caval veins of 70 New Zealand white rabbits. The rabbits were randomly divided into 3 groups according to the different handling methods, including local pLEGFP-N1-tPA transferred group （ gene therapy group ,30 animals ）, pLEGFP-N1 transferred group （ control group, 20 animals ）, DMEM ＋ 10% serum injected group （blank control group,20 animals）. 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 surgery. So we classified the 3 groups into 6 sub-units A1, B1, C1, A2, B2, C2. The EGFP expression of harvested inferior caval veins were observed under the laser confocal microscopy. The thromuses on the surface 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 caval veins of gene therapy group were observed under laser confocal microscopy and there were EGFPs in pLEGFP-N1 group. No EGFPs were seen in blank control group. Seventy Dacron patches plus 10 untransplanted Dacron patches were magnified by stereoscope （ ×160 fold） and electron microscope （ ×500 fold）. It was found that there were no thrombuses on the surface of Dacron patches in untransplanted and gene therapy group and there were many thrombuses on the surface of Dacron patches in control groups. Exogenous tPA bands were found in the inferior caval veins of gene therapy group through Western blot and no exogenous tPA bands in the ve

关 键 词：逆转录病毒科 纤溶酶原 基因治疗 血栓 

分 类 号：R346[医药卫生—基础医学]