纳米粒子介导特异基因转染的实验研究

目的：观察纳米粒子携带特异性基因转染的可行性及其效率,方法：应用聚乳酸聚乙醇酸共聚物和聚乙烯醇包载特异性反义单核细胞趋化蛋白－1基因,制备纳米级粒子混合物。检测其包埋率,体外释放情况及粒度。以阳离子脂质体为对照,利用培养的平滑肌细胞,应用聚合酶链反应（PCR）观察其为真核细胞传递基因的可能性。采用移植静脉内膜增生动物模型,应用RNA斑点杂交和病理形态学分析。观察其在体内的基因转染、表达及生物学效应。结果：制备的纳米粒子－DNA粒度为150－300nm,包埋率为：0．9％,体外释放时间为2周左右。PCR结果提示：纳米粒子可将目的基因转移至平滑肌细胞中,体内实验显示：纳米粒子可实现体内局部基因转染,表达,发挥相应的效应。结论：纳米粒子可用于特异性基因的转染。

Nanoparticle as a new gene transferring vector in specific expression gene

OBJECTIVE: To evaluate the possibility and efficiency of nanoparticle as a new vector in specific gene transference. METHODS: Nanoparticle-DNA complex was prepared with PLGA bearing antisense monocyte chemotactic protein-1 (A-MCP-1), a specific expression gene, and the package efficiency, release progress in vitro, and size of the complex were determined. The nanoparticl-DNA was trasnsfected into the cultured smooth muscle cells. PCR was used to evaluate the transfection of A-MCP-1. Cationic lipid (lipofectamine) was used to transfect A-MCP-1 as control. Forty-eight hours later, DNA in the SMCs was extracted and examined by PCR. Twenty New Zealand White rabbits underwent jugular vein-to-artery bypass grafting procedure, of which 6 received grafts transfected with nanopaticle-A-MCP-1 (200 microgram), 6 received grafts with cationic liposome (DOTAP)-A-MCP-1 (200 microgram), 4 received grafts with LNCX plasmid, and 4 received grafts without transfection as control. Fourteen days after surgery grafts were harvested. The expression of A-MCP-1 and its effect on MCP-1 in vein grafts were detected by dot blotting. The morphology of the grafts was investigated. RESULTS: The package efficiency, release progress in vitro, and size of the nanoparticle-DNA complex thus prepared were 0.9%, 2 week, and 150 nm approximately 300 nm respectively. Genomic DNA PCR showed that A-MCP-1 gene could be successfully transfected into smooth muscle cells by nanoparticle. Two weeks later, antisense MCP-1 was expressed in the vascular walls of the groups with transfection methods by nanaoparticle or by cationic lipid to an almost same degree. The degree of vascular hyperplasia in gene transfection groups was lower than that in control group. There was no significant difference in inhibition of intimal hyperplasia between the two groups of transfection by different vectors. CONCLUSION: Nanoparticle acts as a vector to transfect specific gene in vitro and in vivo.