【作者】 程龙；

【导师】 张凯伦；

【作者基本信息】 华中科技大学 ， 心血管外科， 2012， 博士

【摘要】 第一部分：pIRES-tPA-Dsred Express-2真核表达质粒的构建和体外表达目的：构建携带有组织型纤溶酶原激活因子目的片段的真核表达载体pIRES-tPA-Dsred Express2。验证该质粒转染人脐静脉内皮细胞系EA.hy926是否高表达组织型纤溶酶原激活因子,其产物蛋白是否具有生物学活性。方法：勾取tPA基因的CDS序列,通过基因工程技术将其插入pIRES-Dsred Express2质粒,构建携带有人组织型纤溶酶原激活因子目的片段的真核表达载体pIRES-tPA-Dsred Express2。将构建成功的真核表达载体pIRES-tPA-Dsred Express2用脂质体转染试剂Lipofectamine(?) LTX转染人脐静脉内皮细胞系EA.hy926。应用逆转录实时荧光定量PCR检测转染后48小时,细胞内目的蛋白mRNA含量；Western Blot检测转染后相关蛋白表达情况；应用ELISA技术,检测细胞培养上清中tPA含量；应用酶促反应检测细胞培养上清中人组织型纤溶酶原激活因子活性。观察转染pIRES-tPA-Dsred Express2载体后人脐静脉内皮细胞系EA.hy926对于人组织型纤溶酶原激活因子及其相关蛋白分泌及其活性随时间的变化关系。结果：通过将tPA目的基因片段插入pIRES-Dsred Express2质粒,成功构建了真核表达载体pIRES-tPA-Dsred Express2。体外转染人脐静脉内皮细胞系EA.hy926后48小时可以观察到细胞可激发红色荧光,转染效率为(20.7±4.2)%。逆转录实时定量PCR检测显示pIRES-tPA-Dsred Express2质粒组其tPA, Dsred的mRNA相对含量分别为769.21±35.11及1164.26±82.85,显著高于对照组。Western Blot检测显示,目的蛋白tPA及红色荧光蛋白Dsred含量显著增高。细胞上清人组织型纤溶酶原激活因子含量及人组织型纤溶酶原激活因子活性检测显示pIRES-tPA-Dsred Express2质粒组(4.73±0.02)ng/hour·(105cells),活性为(9.48±0.12)IU/hour-(105cells),显著高于对照组。转染pIRES-tPA-Dsred Express2后内皮细胞上清中的人组织型纤溶酶原激活因子浓度及活性在24小时为最高,而后呈现下降趋势。人组织型纤溶酶原激活因子活性变化受纤溶酶原激活物抑制剂-1影响与其浓度变化方式不同。结论：成功构建了携带有人组织型纤溶酶原激活因子的真核表达载体pIRES-tPA-Dsred Express2。体外转染验证其可正确指导合成及分泌组织型纤溶酶原激活因子,表现出显著纤溶活性,为后续实验奠定了实验基础。第二部分：超声微泡介导pIRES-tPA-Dsred Express2-脂质体复合体体外转染及表达目的：通过超声微泡介导的基因治疗技术将携带有组织型纤溶酶原激活因子基因的质粒pIRES-tPA-DsRed-Express2-脂质体复合体体外转染内皮细胞,使内皮细胞高表达组织型纤溶酶原激活因子,观察超声介导的基因治疗技术对于内皮细胞质粒转染的效果。方法：薄膜水化制备全氟丙烷超声微泡。使用全氟丙烷微泡介导pIRES-tPA-DsRed-Express2-脂质体复合体转染人脐静脉内皮细胞系EA.hy926细胞。通过荧光计数,计算细胞转染效率。应用逆转录实时荧光定量PCR检测转染后48小时,细胞的目的蛋白mRNA含量；应用ELISA技术,检测上清中tPA含量及活性。结果：制备的全氟丙烷超声微泡平均大小为(3.5±1.4)μm。微泡浓度为(3.3±1.2)×108/ml。zeta电位为(-2.2±1.5)mV。制备后的12小时内性质稳定。转染后48小时,超声微泡介导组(UM+LTX)转染效率为(27.3±3.6)%,Lipofectamine(?) LTX转染组(LTX)转染效率为(20.6±2.0)%。逆转录实时荧光定量PCR检测显示UM+LTX组及LTX组,目的蛋白tPA的mRNA的相对含量分别为(953.15±92.77)和(721.32±68.31),具有显著性差异。荧光蛋白Dsred的mRNA相对含量分别为(1191.22±109.31)和(1092.15±102.71)。UM+LTX组其细胞上清中的tPA含量及tPA活性均较LTX显著升高。结论：成功制备了含有全氟丙烷的超声微泡。通过超声微泡介导质粒-脂质体复合体转染内皮细胞EA.hy926,进一步提高了质粒-脂质体复合体转染内皮细胞的转染效率,从而提高目的蛋白组织型纤溶酶原激活因子的表达分泌量,提高了纤溶活性。第三部分：携带可表达tPA真核表达载体PLGA纳米粒-超声微泡复合体的构建及体外吞噬实验目的：构建携带有可表达组织型纤维溶酶激活因子质粒的PLGA纳米粒-超声微泡复合体。检测其理化性质,体外缓释方式,细胞毒效应及其在超声介导下对于细胞摄取纳米粒的影响。方法：应用双次乳化法制备携带有pIRES-tPA-DsRed Express2质粒的PLGA纳米粒；应用薄膜水化超声法制备阳离子超声微泡；两者通过静电吸附的方式形成PLGA纳米粒-超声微泡复合体。检测PLGA纳米粒的载药量,PLGA纳米粒-超声微泡复合体质粒载量；检测纳米粒及纳米粒-超声微泡复合体细胞毒性；检测PLGA纳米粒及PLGA纳米粒-超声微泡复合体体外缓释质粒的方式；检测其在超声辐照介导的微泡解构时介导体外细胞吞噬纳米粒的能力。结果：自制的纳米粒其粒径为(217.2±2.2)nm,zeta电位为(-15.24±0.83)mV。微泡平均大小为(3.2±1.5)μm,Zeta电位为(13.66±2.05)mV。微泡浓度为(4.3±1.1)×108/ml。纳米粒-超声微泡复合体其平均大小为(4.6±1.7)μm。zeta电位为(2.23±1.45)mV。浓度为(3.0±1.3)×108/ml。lmgPLGA纳米粒载有质粒(42.3±2.1)μg。1ml纳米粒-超声微泡复合体中带有质粒(20.5±2.7)μg。纳米粒及纳米粒-超声微泡复合体均表现为在起始段短时内质粒快速释放,后呈现稳定释放的趋势。到达第7天时,释放量达到其总量的(57±3)%。纳米粒及纳米粒-超声微泡复合体均未见明显细胞毒性效应,仅最高浓度组见轻度的细胞活性减少。PLGA纳米粒组及PLGA纳米粒-超声微泡复合体组均可见大量的纳米粒被吞噬。PLGA纳米粒-超声微泡复合体组在超声辐照介导下具有更高的细胞摄取效率。结论：所构建的纳米粒-超声微泡复合体显示出较好的缓释效果,较低的细胞毒性,在超声辐照介导下可增强纳米粒吞噬效率。更多还原

【Abstract】 Part I:Construction of pIRES-tPA-Dsred Express-2Eukaryotic expression vector and expression in vitroObjective To construct a eukaryotic expression vector pIRES-tPA-Dsred Express2which carried the gene of human tissue-type plasminogen activator. Verify the improved expression of tissue-type plasminogen activator while human umbilical vein endothelial cell line EA.hy926were transfected by pIRES-tPA-Dsred Express2. And the biological activity of the product protein was investigated.Method Access to the tPA gene sequence and insert it into the multiple clone sites of pIRES-Dsred Express2. DNA sequencing was used to affirm construction of the recombinant. The vector, pIRES-tPA-Dsred Express2, was transfected into human umbilical vein endothelial cell line EA.hy926cells by Lipofectamine(?) LTX. Reverse transcription real-time fluorescence quantitative PCR was performed to detect mRNA levels at48hours after transfection. Western blot was taken for the target protein expression. Application of ELISA was to assay tPA concentration in the supernatant. The activity of tPA in the supernatant was quantified by enzymatic reactions. The concentration and activity of the secreted tPA versus time relationships was investigated.Results The eukaryotic expression vector pIRES-tPA-Dsred Express2was successfully constructed which verified by DNA sequencing. At48hours after transfection, significantly red fluorescence can be observed in EA.hy926. The transfection efficiency was (20.7±4.2)%. Real-time reverse transcriptase quantitative PCR analysis showed that relative mRNA content of the target protein tPA and red fluorescent protein Dsred in pIRES-tPA-Dsred Express2group was769.21±35.11and1164.26±82.85, significantly higher than that in control. Western Blot showed that tPA and Dsred content was significantly higher in pIRES-tPA-Dsred Express2plasmid group. The concentration and activity of tPA in cell supernatant of pIRES-tPA-Dsred Express2plasmid group was (4.73±0.02)ng/hour-(105cells) and (9.48±0.12) IU/hour(105cells), which is significantly higher than that in control. The concentration and activity of the secreted tPA versus time relationships showed the peak concentration and activity appeared at24hours after transfection. The relationships versus time of tPA activity, the concentration of tPA and PAI-1was associated but different.Conclusion We successfully constructed the eukaryotic expression vector, pIRES-tPA-Dsred Express2, which carrying gene sequence of tissue-type plasminogen activator. And verify that the vector can correctly guide the synthesis and secretion of tissue-type plasminogen activator, showing a significant biological activity in vitro transfection. Part II:In vitro transfection of pIRES-tPA-Dsred Express2-liposome complexes mediated by ultrasound microbubble and expression in endothelial cellsObjective pIRES-tPA-DsRed-Express2-lipidsome complexes was admitted to endothelial cells in vitro mediated by ultrasound microbubble targeted destruction. The effection of ultrasound-mediated gene therapy in the plasmid transfection for endothelial cells was investigated.Method The perfluoropropane ultrasound microbubbles were prepared by thin-film hydration. The pIRES-tPA-DsRed Express2-liposome complexes were transfected into EA.hy926cells mediated by ultrasound microbubble destruction. Cell transfection efficiency, the target protein mRNA relative content in cells, tPA content and activity in the supernatant was detected.Results The average size of perfluoropropane ultrasound microbubbles was (3.5±1.4)μm. The concentration was (3.3±1.2)×108/ml. The zeta potential was (-2.2±1.5) mV. The microbubbles were stable within12hours after preparation. At48hours after transfection, the transfection efficiency of ultrasound microbubble-mediated group (UM+LTX) was (27.3±3.6)%, while (20.6±2.0)%in lipofectamine(?)LTX group (LTX). The relative content of tPA mRNA was respectively (953.15±92.77) and (721.32±68.31) in (UM+LTX) and LTX group. The relative content of fluorescence protein Dsred mRNA was (1191.22±109.31) and (1092.15±102.71) in each group. The concentration and activity of tPA in supernatant of (UM+LTX) group, is significantly higher then that of LTX group.Conclusion The perfluoropropane Ultrasound microbubbles were successfully prepared. Mediated by ultrasound microbubble targeted destruction, the transfection efficiency of the plasmid-liposome complexes for endothelial cells was further improved. Thereby the expression and secretion of tissue-type plasminogen activator was enhanced, as a result the fibrinolytic activity was improved. Part III:Construction of pIRES-tPA-DsRed Express2loaded PLGA nanoparticles-microbubble complexes and in vitro phagocytosis studyObjective To construct pIRES-tPA-DsRed Express2loaded PLGA nanoparticles-ultrasound microbubble complexes. To investigate physical and chemical properties, in vitro plasmid release manner, Cytotoxicity and cellular uptake of nanoparticles-microbubble complexes mediated by ultrasound destruction.Method pIRES-tPA-DsRed Express2loaded PLGA nanoparticles were prepared by double emulsion method. Cationic ultrasound microbubble were prepared by thin-film hydration method. Nanoparticles-microbubble complexes were created by electrostatic adsorption. The plasmid loading of the PLGA nanoparticles and nanoparticles-microbubble complexes was assayed.In vitro release manner and the cytotoxicity of PLGA nanoparticles and nanoparticles-microbubble complexes was Investigated. The phagocytic manner for nanoparticles-microbubble complexes after ultrasound mediated microbubble destruction was studied.Results The size and zeta potential of the plasmid loading PLGA nanoparticles was (217.2±2.2)nm and (-15.24±0.83)mV. The average size and zeta potential of the cationic microbubbles was (3.2±1.5)μm and (13.66±2.05)mV. The concentration is (4.3±1.1)×10/ml. The average size of the nanoparticles-microbubble complexes was (4.6±1.7)μm and zeta potential was (2.23±1.45)mV, and the concentration was (3.0±1.3)×10/ml.1ml nanoparticles-microbubble complexes contained (20.5±2.7)μg plasmid. And PLGA nanoparticles contained (42.3±2.1)μg plasmid per mg. Nanoparticles and nanoparticles-microbubble complexes were both shown a burst release in the initial segment, and then a trend of stable release instead. The total (57±3)%plasmid encapsulated were released in the first seven days. Nanoparticles and nanoparticles-microbubble complexes showed little cytotoxicity. Only the highest concentration group appeared mild reduced cell activity. Nanoparticles-microbubble complexes group showed a higher cellular uptake efficiency after ultrasound mediated microbubble destruction.Conclusion The nanoparticles-microbubble complexes with a good release pattern, low toxicity, enhanced phagocytic efficiency.更多还原