气道内微量雾化机对质粒DNA完整性和基因转染效率的影响

Effect of a New Nebulizer on Plasmid DNA Integrity and Gene Transfection

目的:探索新型气道内微量雾化机对质粒DNA(pDNA)完整性和整体动物基因转染效率的影响。方法:首先,研究新型气道内微量雾化机对pDNA破坏程度。分别向新型气道内微量雾化机和目前临床普遍使用的机械喷射式雾化机的加药池内加入2ml pDNA(20μg/ml),在开始雾化后第1min、3min、5min分别收集两种雾化机嘴处的雾化液滴,用琼脂糖凝胶电泳观察比较质粒的完整性。其次,研究整新型气道内微量雾化机在整体动物上对基因转染效率的影响。分别用新型气道内微量雾化机和目前临床普遍使用的机械喷射式雾化机,给大鼠雾化经多聚乙烯亚胺(polyethylenimine,PEI)修饰的相同量的绿色荧光蛋白质粒(plasmid DNA of green fluorescent protein gene,pEGFP)3.3μg(PEI/pEGFP),24h后提取动物肺组织进行反转录,用real time PCR及琼脂糖凝胶电泳观察分析绿色荧光蛋白(green fluorescent protein,GFP)的mRNA表达情况。结果:新型气道内微量雾化机在雾化第1min、第3min、第5min后完整的质粒比例分别为(99.6±0.7)%、(100±1.2)%、(99.6±0.7)%,与未雾化对照相比(P>0.05,n=3),无统计学差异,新型雾化机对质粒破坏性可以忽略。临床常用的喷射式雾化机在相同时间段内完整的质粒比例分别为(70.3±1.5)%、(49.3±1.5)%、(32.7±0.6)%。与未雾化对照相比(P<0.05,n=3)有统计学差异,并且随雾化时间增加临床常用的喷射式雾化机对质粒的完整性破坏程度逐渐增加;新型气道内微量雾化机的基因转染效率高于临床普遍使用的机械喷射式雾化机转染效率的(382.1±101.1)倍(P<0.01,n=3)。结论:新型气道内微量雾化机对质粒没有破坏性,能显著增加雾化吸入基因转染效率。为雾化基因治疗提供了一种合适的工具。

Objective： Explore the effect of a new airway miniaturized nebulizer on the plasmid DNA（pDNA） integrity and animals gene transfection efficiency.Methods： In the first experiment,the effect of the new airway miniaturized nebulizer on pDNA integrity was tested.2ml pDNA（20μg/ml） was put into the sample pool of the new airway miniaturized nebulizer and a clinical commonly used jet nubulizer respectively.Aerosol samples were collected at the 1st min,3rd min,5th min from the nozzle of the two nebulizers respectively and investigated with agarose gel electrophoresis.In the second experiment,the effect of gene transfection efficiency of the new airway miniaturized nebulizer on animals was studied.3.3μg PEI/pEGFP（polyethylenimine and plasmid DNA of green fluorescent protein complex formation which contain 3.3μg pEGFP） was aerosolized to rats via the new airway miniaturized nebulizer and a clinical commonly used jet nubulizer respectively.Rats were sacrificed 24h after aerosol treatment.The mRNA was abstracted from the lung of those rats,reverse transcribed into cDNA and determined by real-time PCR.PCR products were also investigated with agarose gel electrophoresis.Result： At the 1st min,3rd min,5th min,the proportions of integrity pDNA of the new airway miniaturized nebulizer were（99.6±0.7）%,（100±1.2）%,（99.6±0.7）% respectively,and compared with the control group.The result was no significant difference between the two group（P0.05,n=3）.The effect of the new airway miniaturized nebulizer on plasmid DNA can be neglected.At the 1st min,3rd min,5th min,the proportions of integrity pDNA of the clinical commonly used jet nubulizer were（70.3±1.5）%,（49.3±1.5）%,（32.7±0.6）% respectively,and compared with the control group.The results was significantly different between the two group（P0.05,n=3）.The proportion of integrity pDNA of the jet nubulizer was decline graduately accompany with time.The new nebulizer yielded 382.1±101.1 fold higher expression levels than the ordinary jet nebulizer（P0.01,n=3）Conclution： The new airway miniaturized nebulizer provided an excellent protection on the plasmid DNA,increased the efficiency of gene transfection significantly,and provided a suitable device for aerosol gene therapy.