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| Effect of a New Nebulizer on Plasmid DNA Integrity and Gene Transfection |
| WANG Hong1,2, SUN Wen-wu2, MA Zhuang2 |
1. Department of Respiratory Medicine, Xijing Hospital, Fouth Military Medical University, Xi’an 710032, China;
2. The Army Center of Respiratory and Allergic Disease Diagnosis and Management, General Hospital of Shenyang Military Region, Shenyang 110016, China |
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Abstract 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(P>0.05, 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(P<0.05, 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(P<0.01, 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.
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Received: 28 November 2011
Published: 25 March 2012
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