【作者】 龚永生；

【导师】 张凯伦；

【作者基本信息】 华中科技大学 ， 外科学， 2008， 博士

【摘要】 第一部分pLEGFP-N1-tPA逆转录病毒载体构建、鉴定及包装细胞PT67/pLEGFP-N1-tPA纯系培育目的构建含人组织型纤溶酶原激活剂（tissue-type plasminogen activator, tPA）基因增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）基因逆转录病毒载体及产高滴度病毒的纯包装细胞系。方法用PCR方法扩增目的基因tPA,定向克隆入逆转录病毒载体（pLEGFP-N1）中,酶切反应、PCR及DNA测序鉴定重组逆转录病毒载体pLEGFP-N1-tPA;在基因转染试剂SofastTM介导下将pLEGFP-N1-tPA转入包装细胞PT67,经相应的抗生素筛选、标记、显微移出,培育出全EGFP纯包装细胞系。用NIH3T3细胞测定病毒滴度。结果经限制性酶切分析、DNA序列分析、EGFP的表达证实pLEGFP-N1-tPA中含有tPA基因。转染有pLEGFP-N1-tPA的纯PT67细胞产病毒滴度为1×10⁷CFU/ml。结论成功构建了pLEGFP-N1-tPA载体和产高滴度病毒纯包装细胞系,为tPA局部靶向治疗的理想人工心瓣的研究和应用奠定了实验基础。第二部分pLEGFP-N1-tPA感染ECUV304和心肌细胞,ECUV304/ pLEGFP-N1-tPA建纯系目的观察pLEGFP-N1-tPA感染ECUV304和心肌细胞后,ECUV304/pLEGFP-N1-tPA纯系上清和感染后心肌细胞上清在体外血栓模型中溶栓情况及tPA表达情况。方法pLEGFP-N1-tPA感染ECUV304细胞,单个强表达EGFP的ECUV304细胞→成簇→显微移出→培育→成纯系。建体外血浆平板血栓模型（用人全血）,纯系ECUV304/pLEGFP-N1-tPA培养上清加入模型中,设对照组,观察溶解血栓情况,同时椐蚓激酶活性算出tPA活性。ELISA测出tPA含量。Western blot检测tPA。原代1-4d乳鼠心肌细胞培养,感染pLEGFP-N1-tPA,培养上清加入血浆平板血栓模型,设对照组,观察溶解血栓情况,算出tPA活性,测出tPA含量。Western blot检测tPA。结果转导有pLEGFP-N1-tPA的内皮细胞和心肌细胞培养细胞上清在体外血栓模型中有大的溶解圈。ECUV304/pLEGFP-N1-tPA纯系上清tPA活性是502.16u/10⁶ cells/24h,tPA含量是716.27ng/10⁶ cells/24h,Western blot检出外源tPA条带。心肌细胞/pLEGFP-N1-tPA上清tPA活性是464.27u/10⁶ cells/24h,tPA含量是607.34ng/10⁶ cells/24h,Western blot亦检出外源tPA条带。结论成功建立ECUV304/pLEGFP-N1-tPA纯系,纯系上清有明显溶栓作用,tPA活性及含量均高,有外源tPA条带。心肌细胞/pLEGFP-N1-tPA上清有明显溶栓作用,tPA活性及含量均高,有外源tPA条带。第三部分在体研究特氟隆（Dacron）片移植入兔下腔静脉,Dacron片周围局部转导pLEGFP-N1-tPA目的探讨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2d、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（enzyme linked immunosorbent assay, 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±9.05u/g、537.50±12.45u/g,两时间点比较,差异显著性（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含量无显著差异（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,为研究和应用tPA基因瓣膜奠定了基础。更多还原

【Abstract】 PartⅠConstruction of recombinant enhanced green fluorescent protein gene retroviral vector with tissue-type plasminogen activator gene and pure packaging cell line producing high titer retrovirusesObjective To construct recombinant enhanced green fluorescent protein （EGFP） gene retroviral vector with human tissue-type plasminogen activator （tPA） gene and culture pure packaging cell line producing high titer retroviruses.Methods tPA gene was amplificated by polymerase chain reaction （PCR） and was cloned into retroviral pLEGFP-N1 plasmid. First, the pLEGFP-N1-tPA was identified by restriction endonuclease digestion、PCR analysis and DNA sequencing. Second, the pLEGFP-N1-tPA was transferred into packaging cell PT67 by gene transfection reagent SofastTM. The PT67/ pLEGFP-N1-tPA cells were selected by the corresponding antibiotics. According to the EGFP, the cell expressing strong EGFP was signed. When the signed cell grew as a bundle cells, they were picked out of the plate and cultured to be a pure packaging cell line/ pLEGFP-N1-tPA cells all expressing EGFP. Last, the virus titer produced by pure packaging cells was assayed using NIH3T3.Results The restriction endonuclease digestion, PCR analysis and DNA sequencing confirmed the recombinant pLEGFP-N1-tPA vector containing tPA gene. The virus titer of the pure packaging cell line PT67/pLEGFP-N1-tPA cells was 1×10⁷CFU（cloning forming unit）/ml.Conclusion We have successfully constructed a kind of recombinant retroviral vector pLEGFP-N1-tPA as well as a pure packaging cell line producing high virus titer. This provide a good basis for the study and application of idea synthetic valves with tPA gene local target therapy.PartⅡECUV304 and heart myocytes were infected by pLEGFP-N1-tPA and the purified ECUV304/pLEGFP-N1-tPA cell line was constructed.Objective To observe thrombolysis of supernatant from purified ECUV304/ pLEGFP-N1-tPA cell line and infected cardio myocytes in vitro thrombi model and tPA expressing.Methods ECUV304 was infected by pLEGFP-N1-tPA and one ECUV304/ pLEGFP-N1-tPA cell expressing strong EGFP was cultured as a bundle. Bundle of ECUV304/pLEGFP-N1-tPA→moved out plate by micro operation to culture as a cell line. Supernatant from purified ECUV304/pLEGFP-N1-tPA cell line with contrast were dropped into vitro thrombi model and thrombolysis were observed. According to the activity of lumbrukinase, tPA activity was accounted. tPA content was measured by ELISA. Western blot checked out exogenous tPA band. Origin cardio myocytes from 1-4d neonate rats were infected by pLEGFP-N1-tPA. tPA was checked as above.Results Supernatant from purified ECUV304/pLEGFP-N1-tPA cell line and infected cardio myocytes had obviously big thrombolysis zone. tPA activity from purified ECUV304/pLEGFP-N1-tPA cell line was 502.16u/10⁶ cells/24h and content of tPA was 716.27ng/10⁶ cells/24 h. Exogenous tPA band was checked out by Western blot. tPA activity from infected cardio myocytes was 464.27u/10⁶ cells/24h and content of tPA was 607.34ng/10⁶ cells/24h. As the same condition, exogenous tPA band was checked out by Western blot.Conclusion We successfully cultured purified ECUV304/pLEGFP-N1-tPA cell line and supernatant from that cell line had obviously thrombolysis. Activity and content of tPA were all high and exogenous tPA band was checked out. Also, supernatant from infected heart muscle cells by pLEGFP-N1-tPA had apparently thrombolysis zone. Activity and content of were high and exogenous tPA band were demonstrated.PartⅢObserving thrombolysis on exogenous Dacron patches transplanted in inferior caval vein that transferred pLEGFP- N1-tPA in localObjective To identify the target thrombolysis of recombinant tissue-type plasminogen activator （tPA） transferred into the tissue around the Dacron patch （the same materials making of the ring of mechanical valve） in inferior caval vein in vivo.Methods 70 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 confocal. The thrombi on the surface of Dacron patches were detected by stereoscope and electron microscope. The tPA expression in inferior caval veins were detected by Western blot and their thrombolysis and activities were observed and calculated in plasma plates. ElISA were used to identify the contents of tPA.Results At the second, 75th day, much and strong EGFPs in inferior caval veins of gene therapy group were observed under confocal and there were EGFPs in pLEGFP-N1 group. No EGFPs were seen in blank control group. 70 Dacron patches plus 10 untransplanted Dacron patches were magnified by stereoscope （×160 fold） and electron microscope （×500 fold）. We found that there were no thrombi on the surface of untransplanted and gene therapy group Dacron patches and there were many thrombi on the surface of control groups Dacron patches. Exogenous tPA bands were found in the inferior caval veins of gene therapy group through Western blot and no exogenous tPA bands in the veins of control groups. According to the plasma plates, big areas of thrombolysis were found in the plasma plates drop by substances from gene therapy group veins and this indicated strong thrombolysis. On the contrary, there were no thrombolysis in control groups. According to the activity of lumbrukinase, we counted that tPA activities from gene therapy group was 529.62±9.05u/g and 537.50±12.45u/g respectively on 2nd and 75th day after operation. The activities of tPA there were no significant variation between 2nd and 75th （P>0.05）. The tPA contents of inferior caval veins from 6 sub-units were （737.64±13.19）、（29.88±5.61）、（28.7±5.49）、（742.87±10.56）、（32.03±6.26）、（31.34±5.63） ng/g corresponding to A₁、B₁、C₁、A₂、B₂、C₂. Contents of tPA in gene therapy were obviously higher than that in control groups （P<0.01）. tPA contents there were no significant difference between 2nd and 75th day in gene therapy group （P>0.05）.Conclusion pLEGFP-N1-tPA locally transferring can effectively prevent thrombosis on the surface of exogenous transplant. This provide a deeply basis for the study and application of tPA gene valve.更多还原