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目的观察纳米粒子介导血管内皮生长因子(VEGF)基因转染的可行性,了解心肌缺血动物模型经心肌直接注射VEGF基因纳米粒子后新生血管以及心功能改善情况。方法应用聚乳酸聚乙醇酸共聚物(PLGA)和聚乙烯醇(PVA)包载VEGF165基因质粒,制备纳米级粒子混合物,检测其载药量、体外释放情况及粒径;培养乳鼠心肌细胞,转染VEGF165基因纳米粒子(VEGF纳米粒子),应用RT-PCR法检测VEGF mRNA水平,ELISA法检测VEGF蛋白表达水平;将VEGF纳米粒子悬浮液注射至活体家兔心肌内,96h后心肌注射部位取材,电镜观察其向心肌组织内传递基因的可行性;建造兔心肌缺血模型,分别将VEGF纳米粒子(12只)、VEGF165裸质粒(12只)或生理盐水(对照组,8只)注射至缺血部位心肌,1个月后心脏超声监测,然后处死,组织学切片观察心肌组织中毛细血管生成情况。结果制备的纳米粒子载药量为1·87%,粒径为50~300nm;RT-PCR和ELISA结果提示纳米粒子可将目的基因转移至培养的心肌细胞中;体内实验显示心肌细胞胞质内及胞核内可见大量被吞噬的纳米粒子;术后1个月超声检查显示,VEGF纳米粒子组室壁运动幅度(1·87mm±0·32mm)、左室射血分数(60%±10%)较裸质粒组(1·59mm±0·24mm,50%±6%)及对照组(0·93mm±0·40mm,40%±8%)改善更加明显,各组之间差异均有统计学意义(均P<0·05);组织学检查显示,在100倍光镜视野下心肌内毛细血管数VEGF纳米粒子组(57个±12个)明显高于裸质粒组(41个±14个)及对照组(24个±8个),各组之间差异有统计学意义(均P<0·05)。结论纳米粒子可作为VEGF基因向心肌组织转运的载体,在兔心肌缺血模型经心肌直接注射VEGF165基因纳米粒子,能够促进心肌内毛细血管新生,达到改善心功能的目的。
Objective To observe the feasibility of nanoparticle-mediated transfection of vascular endothelial growth factor (VEGF) gene and to understand the angiogenesis and cardiac function of myocardium after injection of VEGF gene nanoparticles directly into myocardium. Methods Polylactic acid polyglycolic acid copolymer (PLGA) and polyvinylalcohol (PVA) were used to encapsulate the VEGF165 gene plasmid to prepare nanometer particle mixture. The drug loading, release in vitro and particle size were measured. The cultured neonatal rat cardiomyocytes, The VEGF165 gene nanoparticles (VEGF nanoparticles) were transfected, VEGF mRNA was detected by RT-PCR and VEGF protein by ELISA. The suspension of VEGF nanoparticles was injected into the myocardium of living rabbits. (12 mice), 12 naked VEGF165 mice or normal saline (control group, 8 mice) were injected into the myocardium of rabbits to establish a model of myocardial ischemia. Myocardial ischemia, 1 month after the heart ultrasound monitoring, and then sacrificed, histological sections were observed in myocardial tissue capillary angiogenesis. Results The drug loading of the prepared nanoparticles was 1.87% and the particle size was 50-300 nm. The results of RT-PCR and ELISA suggested that the nanoparticles could transfer the target gene into cultured cardiomyocytes. In vivo experiments showed that the intracellular And a large number of nanoparticles were swallowed in the nucleus. Ultrasonography showed that the amplitude of wall motion (1.87mm ± 0.32mm), left ventricular ejection fraction (60% ± 10% ) Was more obvious than that of naked plasmid group (1.59mm ± 0.24mm, 50% ± 6%) and control group (0.93mm ± 0.40mm, 40% ± 8%), with statistical differences among the groups (P <0.05). Histological examination showed that the numbers of intramyocardial vascular endothelial growth factor (VEGF) nanoparticles (57 ± 12) in the 100 × light microscope were significantly higher than those in the naked plasmid group (41 ± 14 ) And control group (24 ± 8), the difference between each group was statistically significant (P <0.05). CONCLUSIONS: Nanoparticles can be used as carriers of VEGF gene transport to myocardial tissue. In the rabbit model of myocardial ischemia, VEGF165 gene nanoparticles can be directly injected into myocardium to promote capillary angiogenesis and improve cardiac function.