功能化Fe_3O_4的制备及在基因转染中的应用

Preparation and application of functionalized DMNP nanoparticles as gene carrier

目的探讨修饰了多聚赖氨酸的超顺磁性葡聚糖Fe3O4纳米粒子(简称功能化Fe3O4,DMNP)的制备及其作为基因载体的可行性。方法采用碱沉淀法一步合成了外包葡聚糖的磁性纳米粒子,并用多聚赖氨酸对其表面进行修饰,使之通过静电作用吸附连接DNA。同时应用扫描电镜、红外分光光度计对该纳米复合物的结构及成分进行表征,并用琼脂糖凝胶电泳和紫外分光光度计对其结合质粒DNA的能力进行测量,该复合纳米粒子作为基因载体将绿色荧光蛋白质粒pEGFP-C1转入人肺癌细胞系A549中,并进一步将载肺癌耐药基因ABCG2-PCDNA3.1质粒转入人肺癌细胞系A549中。结果该复合纳米粒子分散性好,大小较均一,与质粒DNA有较好的结合能力,用荧光显微镜观察到了绿色荧光蛋白的表达,并用PCR技术检测到了耐药基因ABCG2的表达。结论该复合纳米粒子能作为一种新型有效的基因载体在体外将载肺癌耐药基因ABCG2-PCDNA3.1质粒转入人肺癌细胞系A549中并表达。

Objective To explore the preparation of superparamagnetic dextran iron oxide nanoparticles （DMNP） modified with poly-L-lysine, and the feasibility of DMNP used as gene carrier in vitro. Methods Dextran coated magnetic iron oxide nanoparticles were composited by developing a convenient and one-step deposition route. Then the compound nanoparticles were modified with poly-L-lysine on the surface. These particles （DMNP） combined with plasmid DNA by static electrical charges. The characters ofthe nanoparticles were explored by several methods including scanning electron microscopy（SEM）, fourier transform infrared spectroscopy （FTIR） spectra. The DMNP＇s potential of combination with plasmid DNA was also analyzed by electrophoresis and UV. The DMNP bound with plasmid pEGFP-C 1 as one of gene carriers was transfected into human lung cancer cell line A549. The plasmid which carried the drug resistance gene of ABCG2-PCDNA3.1 was also transfected into these cells. Results These compound nanoparticles were very uniform and dispersed homogeneously. It was found that they were very good carriers for plasmid DNA. The expression of green fluorescent protein were observed by inverted microscope. And the expressions of drug resistance gene ABCG2 were detected successfully by polymerase chain reaction （PCR）. Conclusion The nanoparticle is one kind of novel and effective gene cartier. It can transfect the plasmid which carries the drug resistance gene of ABCG2-PCDNA3.1 into lung cancer cell line A549 in vitro, And the gene has been expressed in the cells.