携带tPA真核表达载体PLGA纳米粒-超声微泡复合体的构建及体外实验研究

发布时间：2018-05-11 11:43

本文选题：组织型纤溶酶原激活因子 + 抗凝治疗　；参考：《华中科技大学》2012年博士论文

【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。
[Abstract]:Part one: Construction and expression in vitro of pIRES-tPA-Dsred Express-2 eukaryotic expression plasmid
Objective: to construct a eukaryotic expression vector pIRES-tPA-Dsred Express2. carrying the target fragment of the tissue type plasminogen activator, to verify whether the plasmid transfected to the human umbilical vein endothelial cell line EA.hy926 is highly expressed as a tissue type plasminogen activator, and whether its product protein has bioactivity.
Methods: to draw the CDS sequence of tPA gene and insert it into pIRES-Dsred Express2 plasmid by gene engineering technology to construct the eukaryotic expression vector pIRES-tPA-Dsred Express2. carrying human tissue type plasminogen activator, pIRES-tPA-Dsred Express2. will construct a successful eukaryotic expression vector pIRES-tPA-Dsred Express2 with liposome transfection reagent Lipofe Ctamine (?) LTX transfection of human umbilical vein endothelial cell line EA.hy926. was used to detect the content of target protein mRNA in cells after 48 hours transfection by reverse transcription real-time quantitative PCR; Western Blot was used to detect the expression of related protein after transfection; tPA content in cell culture was detected by ELISA technology and cell culture was detected by enzymatic reaction. The activity of human tissue type plasminogen activator in the supernatant was observed. The relationship between the secretion and activity of human tissue type plasminogen activator and related proteins and its activity with time was observed after the transfection of pIRES-tPA-Dsred Express2 vector EA.hy926 in human umbilical vein endothelial cell line.
Results: by inserting the tPA target gene fragment into the pIRES-Dsred Express2 plasmid, the eukaryotic expression vector, pIRES-tPA-Dsred Express2., was successfully constructed for 48 hours after transfection of the human umbilical vein endothelial cell line EA.hy926, and the cell activation red fluorescence was observed. The transfection efficiency was (20.7 + 4.2)%. Reverse transcriptase real-time quantitative PCR detection showed pIRE. The relative content of mRNA in the S-tPA-Dsred Express2 plasmid group was 769.21 + 35.11 and 1164.26 + 82.85 respectively, which was significantly higher than the.Western Blot detection in the control group. The content of the target protein tPA and the red fluorescent protein Dsred content was significantly increased. The content of the fibrinolytic plasminogen activator in the human cell supernatant and the activation of the human tissue type plasminogen Subactivity detection showed that the pIRES-tPA-Dsred Express2 plasmid group (4.73 + 0.02) ng/hour / (105cells), the activity was (9.48 + 0.12) IU/hour- (105cells), significantly higher than the control group. The concentration and activity of human tissue plasminogen activator in the endothelial cell supernatant after transfection of pIRES-tPA-Dsred Express2 was the highest in 24 hours, and then decreased. The activity of tissue type plasminogen activator is affected by plasminogen activator inhibitor -1 and its concentration varies.
Conclusion: the eukaryotic expression vector pIRES-tPA-Dsred Express2. carrying human tissue plasminogen activator was successfully constructed and transfected in vitro to prove that it can correctly guide the synthesis and secretion of tissue type plasminogen activator, showing significant fibrinolytic activity, which provides an experimental basis for subsequent experiments.
The second part: ultrasound microbubbles mediated transfection and expression of pIRES-tPA-Dsred Express2- liposome complex in vitro.
Objective: to transfect the plasmid pIRES-tPA-DsRed-Express2- liposome complex with the tissue type plasminogen activator gene into endothelial cells in vitro by ultrasound microbubble mediated gene therapy, and to make endothelial cells highly expressed tissue plasminogen activator and observe the hyper mediated gene therapy for endothelial cells. The effect of plasmid transfection.
Methods: perfluoropropane ultrasound microbubbles were prepared by hydrofluorescence. The human umbilical vein endothelial cell line EA.hy926 cells were transfected with perfluoropropane microbubbles mediated pIRES-tPA-DsRed-Express2- liposome complex. The transfection efficiency was calculated by fluorescence count. The target protein M was detected by RT real-time quantitative PCR for 48 hours after transfection. RNA content; ELISA technology was applied to detect the content and activity of tPA in supernatant.
Results: the average size of the prepared perfluoropropane microbubbles was (3.5 + 1.4) microbubbles (3.3 + 1.2) and 108/ml.zeta potential was (-2.2 + 1.5) mV. for 12 hours. The transfection efficiency of the ultrasound microbubble mediating group (UM+LTX) was (27.3 + 3.6)% after 48 hours transfection, and the transfection efficiency of Lipofectamine (?) LTX transfection group (LTX) was (20.6 +). 2) the reverse transcriptase real-time quantitative PCR detection showed that the relative content of mRNA in the target protein tPA was (953.15 + 92.77) and (721.32 + 68.31), respectively. The relative content of the mRNA of the fluorescent protein Dsred was (1191.22 + 109.31) and the tPA content and tPA in the cell supernatant of (1092.15 + 102.71).UM+LTX group respectively. The activity was significantly higher than that of LTX.
Conclusion: the ultrasound microbubbles containing perfluoropropane were successfully prepared. The transfection of the endothelial cell EA.hy926 by the ultrasound microbubble mediated plasmid liposome complex could further improve the transfection efficiency of the plasmid liposome complex transfected to the endothelial cells, thus improving the expression and secretion of the fibrinogen activator of the target protein tissue, and improving the fibrinogen. Dissolve activity.
The third part: Construction of tPA carrying eukaryotic expression vector PLGA nanoparticle ultrasound microbubble complex and its phagocytosis in vitro.
Objective: to construct a PLGA nanoparticle - ultrasonic microbubble complex carrying plasmids which can express tissue type fibrinolytic enzyme activator, and to detect its physicochemical properties, in vitro release mode, cytotoxic effect and its effect on cell uptake nanoparticles under ultrasound.
Methods: the PLGA nanoparticles carrying pIRES-tPA-DsRed Express2 plasmid were prepared by the double emulsification method, and the cationic ultrasonic microbubbles were prepared by the membrane hydration ultrasonic method, and the PLGA nanoparticles ultrasonic microbubble complex was formed by electrostatic adsorption. The drug loading of PLGA nanoparticles and the PLGA nanoparticles ultrasonic microbubble complex plasmid were detected. Loading; detection of cytotoxicity of nanoparticles and nanoparticles - ultrasonic microbubble complexes; detection of PLGA nanoparticles and PLGA nanoparticles - ultrasonic microbubble complex in vitro release plasmids; the ability to detect the phagocytosis of nanoparticles during ultrasound mediated microbubble deconstruction.
Results: the particle size of the homemade nanoparticles is (217.2 + 2.2) nm, the average zeta potential is (-15.24 + 0.83) mV. microbubbles (3.2 + 1.5) mu m, Zeta potential is (13.66 + 2.05) mV. microbubble concentration (4.3 + 1.1) x 108/ml. nanoparticles - the average size of (4.6 + 1.7) mu m.zeta potential is (2.23 + 1.45) mV. concentration (2.23 + 1.45) mV. concentration L.lmgPLGA nanoparticles loaded with plasmid (42.3 + 2.1) mu g.1ml nanoparticles with plasmid (20.5 + 2.7) Mu g. nanoparticles and nanoparticle ultrasonic microbubble complex all showed a rapid release in the initial stage of endoplasmic reticulum, and then showed a stable release trend. The release amount reached (57 + 3)% of the total amount of nanoparticles at seventh days. No obvious cytotoxic effect was found in the nanoparticle - ultrasound microbubble complex, but only the highest concentration group showed that a large number of nanoparticles were phagocytic.PLGA nanoparticles - ultrasonic microbubble complex group with a higher cell uptake in the.PLGA nanoparticles group and the PLGA nanoparticles - ultrasonic microbubble complex group. Take efficiency.
Conclusion: the constructed nanoparticles - ultrasonic microbubble complex showed better sustained release effect and lower cytotoxicity, which could enhance the phagocytosis efficiency of nanoparticles under ultrasound irradiation.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

【相似文献】