机械喷射式雾化机对质粒DNA完整性的影响及保护性研究

目的:探索目前临床广泛使用的喷射式雾化机对质粒DNA(pDNA)完整性破坏的影响及保护措施。方法:在研究雾化时间对pDNA完整性影响的实验中,加入5ml pDNA(20μg/ml),分别在开始雾化后收集第1min内;第2min内;第3min内;第4min内;第5min内及第10min内雾化器喷口处的雾化液滴;在研究加样量对裸pDNA完整性影响的实验中,分别取2ml、4ml、6ml、8ml,各雾化4min,收集最后一分钟内雾化液滴。在两种高分子聚合物对pDNA保护性研究的实验中,各取4ml未经高分子聚合物修饰的以及经过聚乙烯亚胺(polyethylenimine, PEI)或阳离子脂质体修饰后的pDNA,分别雾化10min,收集最后一分钟内雾化液滴。使用琼脂糖凝胶电泳分析雾化样本质粒完整性。结果:在雾化时间由1min增至10min后完整性部分所占百分比由(83.5±2.2)%降至(37.1±2.8)%;加样量由2ml 增至8ml 后,pDNA完整性部分所占百分比由(32.1±3.5)%增至(93.6±0.6)%;经过PEI或阳离子脂质体修饰后的pDNA在雾化过程中几乎无破坏现象。结论:喷射式雾化机对pDNA的破坏呈剂量、时间依赖性,PEI与阳离子脂质体对pDNA保护效果良好,为喷射式雾化机在基因雾化治疗中的应用打下一定基础。

Effect of Jet Nebulizer on Plasmid DNA Integrity and the Research of Protective Methods

Objective: To explore the effect of jet nebulizer on plasmid DNA (pDNA) integrity and the protective methods. Methods: In the experiment to determine the effect of nebulization time on pDNA integrity, 5ml pDNA solution was aerosolized. Aerosol samples were collected in the 1^st min, 2^nd min, 3^rd min, 4^th min, 5^th min and the 10^th min. In the experiment to determine the effect of aerosolized solution amount on pDNA integrity, 2 ml、4 ml、6 ml、8ml pDNA solution was aerosolized for 4 min respectively. Aerosol samples were collected in the last minute. In the experiment to determine the protective effectiveness of the two polyplex, 4 ml naked pDNA solution, 4ml PEI-DNA solution and 4 ml liposome-DNA solution was aerosolized for 10 min respectively. Aerosol samples were collected in the last minute. Degradation associated with the aerosolisation process was investigated with agarose gel electrophoresis. Results: As aerosolization time increased from 1min to 10min, the intact pDNA ratio decreased from (83.5±2.2)% to (37.1±2.8)%. As the pDNA solution amount added from 2 ml up to 8ml, the intact DNA ratio ascended from (32.1±3.5)% to (93.6±0.6)%. There was almost no damage to the PEI or cationic lipid protected pDNA solution. Conclusion: The damage to which jet nebulizer exerted on pDNA was related with time and dosage. PEI and cationic lipids provided plasmid DNA with excellent protection, which laid a good foundation for the application of jet nebulizer in future gene aerosol treatment.