包裹E1A基因的纳米粒子制备及转染人肺腺癌细胞A549实验研究

Transfection of Human Pulmonary Adenocarcinoma Cell A549 with Nanoparticles Containing E1A Gene

目的制备包裹E1A基因(腺病毒早期表达基因)的纳米粒子,并观察其介导E1A基因转染人肺腺癌细胞A549的可行性和效率。方法应用聚乳酸聚乙醇酸共聚物和聚乙烯醇包载E1A基因,制备纳米级粒子混合物,检测其包埋率、体外释放情况及粒径大小。用制备的包裹DNA纳米粒子转染人肺腺癌细胞A549,并以阳离子脂质体为对照,用PCR、RT-PCR方法分别检测转染细胞中E1A基因DNA整合和mRNA表达。结果制备的纳米粒子粒径为150～280nm,包埋率为0.78%,体外释放约为22d;在转染相等质量的DNA情况下,纳米组所得克隆数较脂质体组多(P<0.05);PCR、RT-PCR结果表明纳米粒子和脂质体转染细胞均有E1A基因整合和mRNA表达。结论成功制备了纳米粒子,纳米粒子可携带外源基因进行基因转染。

Objective To prepare E1A gene containing nanoparticles and to determine the efficiency of transfection in human pulmonary adenocarcinoma cell A549. Method Nanoparticle-DNA complex with PLGA and adenoviral early expression gene （E1A） was prepared. The efficiency of packaging, in vitro release rate, and size of the complex were determined. The nanoparticle-DNA was transfected into human pulmonary adenocarcinoma cell A549. Cationic lipid （lipofectamine） was used as control. PCR and RT-PCR were used to determine the integration of E1A gene and mRNA expression in transfected cells. Result The packaging efficiency, release rate, and the size of the nanoparticle-DNA complex was 7.8%, 22 days, and 150-280 nm, respectively. More positive transgene cell clones was observed in cells transfected with nanoparticle-DNA than the cells with lipofectamine-DNA （P〈0.05）. Conclusion A high transfection efficiency of A549 cells is achieved with nanoparticle-DNA complex.