新型纳米磁性材料纯化血管内皮生长因子质粒的研究(英文)

A study on purifying of vascular endothelia growth factor plasmid by a new type of magnetic nanoparticles

背景:血管内皮生长因子(VEGF)是基因治疗下肢多平面、多节段、小动脉粥样硬化闭塞症的最常用的治疗基因,基因治疗需要高纯度和大剂量的质粒,而目前的质粒纯化方法存在着成本高、操作繁琐、接触有毒试剂等缺点。目的:探索一种新的方法提纯VEGF质粒。设计:对照实验研究。地点和对象:实验由上海第二医科大学附属仁济医院血管外科完成。对象为磁性纳米粒子(华东理工大学超细粉末研究所惠赠)。干预:制备和修饰3种磁性纳米粒子:二乙基氨基乙基直接连接的磁性纳米粒子(DEAE-MNP),三链螺旋结构的磁性纳米粒子(TMNP)和探针直接连接的磁性纳米粒子(PMNP),分别从细菌裂解上清液中提纯VEGF质粒。主要观测指标:紫外分光光度计检测吸光率(A值)及纯度、琼脂糖凝胶电泳、目的基因测序。结果:①3种磁性纳米粒子均能从细菌裂解上清液中提取VEGF质粒,A值分别为:DEAE-MNP,0.1160±0.0071;TMNP,0.01825±0.0038;PMNP,0.01852±0.0012;纯度与层析柱法(金标准)无统计学差异,其中DEAE-MNP的得率最高。②电泳图像中未见杂质及其他核酸。③质粒送交测序,目的基因序列与文献报道相符。结论:该方法为从混合物中提取高纯度的生物活性物质开创了新途径。

BACKGROUND:Vascular endothelia growth factor(VEGF) is a most common therapeutic gene applied in the treatments of small atherosclerosis obliterans in multiple segments and at multiple planes in lower limbs. Gene therapy requires high purity and quantity of plasmids; however, there are some shortcomings in the existing plasmids purifying methods, such as, high cost, fussy operation, toxic reagent contact, etc.. OBJECTIVE:To explore a new method for purifying VEGF plasmids. DESIGN:A controlled experimental study was conducted. SETTINGS and PARTICIPANTS:Study was completed in the Department of Vascular Surgery,Renji Hospital,Second Medical University. Magnetic nanoparticles were presents from Superfine Powder Institute of East China University of Science and Technology. INTERVENTION:Preparation and modification of three magnetic nanoparticles: DEAE coupled directly to magnetic nanoparticles (DEAE MNP), triplex magnetic nanoparticles (TMNP), and probe coupled directly to the magnetic nanoparticles (PMNP). VEGF plasmids were purified from supernatant fluids of bacterial lysis respectively. MAIN OUTCOME MEASURES:Ultraviolet spectrophotometer absorbance (A) and purity, agaroses gel electrophoresis, and objective gene sequencing. RESULTS: ①VGEF plasmids could be purified from the supernatant of bacterial lysis by all three magnetic napoparticles. The A values for these three nanoparticels were 0.116 0±0.007 1 of DEAE MNP, 0.018 25±0.003 8 of TMNP and 0.0182 5±0.001 2 of PMNP respectively. There was no significant difference in purity compared with that of chromatography (gold standard), in which the DEAE MNP had the highest rate in the abstraction.②No impurity and other nucleic acid were seen in the images of electrophoresis.③Plasmid was sent for sequencing, which the objective gene sequences were accordance with the results of literatures. CONCLUSION:Our technique develops a new approach in the abstraction of bioactive substances with a high purity from mixtures.