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：2018-08-14 17:30

【摘要】：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-DsredExpress2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。WesternBlot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。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。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。1mgPLGA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]:AIM: To construct an eukaryotic expression vector pIRES-tPA-Dsred Express2 carrying the target fragment of tissue plasminogen activator (TPA). To verify whether the plasmid transfected human umbilical vein endothelial cell line EA.hy926 overexpresses TPA and whether the product protein has biological activity.
METHODS: CDS sequence of tPA gene was cloned and inserted into pIRES-Dsred Express2 plasmid by genetic engineering technology to construct eukaryotic expression vector pIRES-tPA-Dsred Express2 carrying the target fragment of human tissue plasminogen activator. Tamine LTX transfected human umbilical vein endothelial cell line EA.hy926.Reverse transcription real-time fluorescence quantitative PCR was used to detect the content of target protein mRNA 48 hours after transfection; Western Blot was used to detect the expression of related protein after transfection; ELISA was used to detect the content of tPA in cell culture supernatant; and enzyme-catalyzed reaction was used to detect the content of tPA in cell culture supernatant. The secretion and activity of human tissue plasminogen activator and its related proteins by human umbilical vein endothelial cell line EA.hy926 after transfection with pIRES-tPA-Dsred Express2 vector were observed.
Results: The eukaryotic expression vector pIRES-tPA-Dsred Express2 was successfully constructed by inserting the target gene fragment of tPA into pIRES-Dsred Express2 plasmid. After transfection into human umbilical vein endothelial cell line EA.hy926 in vitro, red fluorescence was observed 48 hours after transfection, and the transfection efficiency was (20.7 4.2)%. Reversed transcription real-time quantitative PCR showed that pIRE could be transfected into human umbilical vein Endo The relative contents of tPA and Dsred mRNA in S-tPA-Dsred Express 2 plasmid group were 769.21 (+ 35.11) and 1164.26 (+ 82.85) respectively, which were significantly higher than those in the control group. Western Blot assay showed that the contents of tPA and red fluorescent protein Dsred were significantly increased. The activity of pIRES-tPA-Dsred Express2 plasmid group (4.73.02) ng/hour ((105 cells) was significantly higher than that of control group (9.48.12) IU/hour ((105 cells). After transfection of pIRES-tPA-Dsred Express2, the concentration and activity of human tissue plasminogen activator in the supernatant of endothelial cells were the highest at 24 hours, and then decreased. The change of human tissue plasminogen activator activity is affected by plasminogen activator inhibitor-1 in a different way than its concentration.
CONCLUSION: The eukaryotic expression vector pIRES-tPA-Dsred Express2 carrying human tissue plasminogen activator was successfully constructed. The results of in vitro transfection showed that it could guide the synthesis and secretion of tissue plasminogen activator and showed significant fibrinolytic activity, which laid the experimental foundation for the follow-up experiments.
Objective: To investigate the effect of ultrasound-mediated gene therapy on endothelial cells by transfecting plasmid pIRES-tPA-DsRed-Express2-liposome complex carrying tissue plasminogen activator gene into endothelial cells. Plasmid transfection.
METHODS: Perfluoropropane ultrasound microbubbles were prepared by membrane hydration. The pIRES-tPA-DsRed-Express2-liposome complex was used to transfect human umbilical vein endothelial cell line EA.hy926 cells. RNA content; ELISA technology was applied to detect the content and activity of tPA in supernatant.
Results: The average size of perfluoropropane ultrasound microbubbles was (3.5 +1.4) micron. The concentration of microbubbles was (3.3 +1.2)*108/ml. Zeta potential was (-2.2 +1.5) mV. The transfection efficiency was (27.3 +3.6)% in UM + LTX group and (20.6 +2.0)% in Lipofectamine LTX transfection group within 12 hours after transfection. Reverse transcription real-time fluorescence quantitative PCR assay showed that the relative content of tPA mRNA in the supernatant of UM+LTX group and LTX group was (953.15+92.77) and (721.32+68.31), respectively. The relative content of fluorescent protein Dsred mRNA was (1191.22+109.31) and (1092.15+102.71), respectively. Sex was significantly higher than that of LTX.
CONCLUSION: Perfluoropropane-containing ultrasound microbubbles were successfully prepared. The transfection efficiency of plasmid-liposome complex into endothelial cells EA.hy926 was further improved by ultrasound microbubbles, and the expression and secretion of tissue-type plasminogen activator (TIPA) of the target protein were increased. Dissolve activity.
OBJECTIVE: To construct PLGA nanoparticles-ultrasound microbubble complexes with plasmids expressing tissue-type plasmin activator (TIPA) and to investigate its physicochemical properties, in vitro sustained release mode, cytotoxic effect and its effect on cell uptake of nanoparticles under ultrasound.
METHODS: PLGA nanoparticles with plasmid pIRES-tPA-DsRed Express 2 were prepared by double emulsification method, cationic ultrasound microbubbles were prepared by film hydration ultrasonic method, and PLGA nanoparticles-ultrasound microbubbles were formed by electrostatic adsorption. The drug loading of PLGA nanoparticles and PLGA nanoparticles-ultrasound microbubbles composite particles were measured. The cytotoxicity of nanoparticles and nanoparticles-ultrasound microbubble complex, the mode of slow-release plasmid of PLGA nanoparticles and PLGA nanoparticles-ultrasound microbubble complex in vitro, and the ability of extracellular cells to phagocytosis nanoparticles during ultrasound-induced microbubble decomposition were examined.
Results: The size of the nanoparticles was 217.2 (+ 2.2) nm, the zeta potential was (- 15.24 (+ 0.83) mV. The average size of the microbubbles was (3.2 (+ 1.5) microbubbles, and the Zeta potential was (13.66 (+ 2.05) mV. The concentration of the microbubbles was (4.3 (+ 1.1) * 108 / ml. The average size of the nanoparticles-ultrasonic microbubbles was (4.6 (+ 1.7) microbubbles. 1 mg PLGA nanoparticles were loaded with plasmids (42.3 There was no significant cytotoxic effect in both nanoparticles and ultrasound microbubble complexes, but only slight decrease in cell activity in the highest concentration group. A large number of nanoparticles were phagocytized in both PLGA nanoparticles and PLGA nanoparticles-ultrasound microbubble complexes. PLGA nanoparticles-ultrasound microbubble complexes had higher cell uptake under ultrasound irradiation. Take efficiency.
CONCLUSION: The nanoparticles-ultrasound microbubbles composite exhibits better sustained release effect and lower cytotoxicity, and can enhance the phagocytic 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】：R392

【共引文献】