载报告基因壳聚糖纳米粒制剂体外转染活性考察

Test the Transfection Activity of DNA-chitosan Nanoparticles Preparation

目的 考察壳聚糖纳米粒体外基因转染活性及壳聚糖纳米粒经表面修饰的体外转染活性变化。方法 用复凝聚法制备纳米粒 ,透射电镜观察粒子形态 ,体外基因转染实验评价纳米粒的体外转染活性 ,用倒置荧光显微镜观察和流式细胞仪测定转染结果。通过考察递送不同剂量的基因和转染后不同时间的转染效率 ,寻找本递送系统较好的转染条件。用纳米粒表面连接PEG以及半乳糖基白蛋白 ,对纳米粒进行修饰 ,通过体外转染实验 ,评价表面修饰对其转染活性的影响。结果 未经表面修饰的载基因纳米粒能够转染人胚胎肾细胞 (HEK2 93)和肝癌细胞 (HepG2 ) ,但转染效率仍不如脂质体转染试剂 ,且在转染实验后约 72h较高 ,递送基因较佳的剂量是 4 μg ,在以上两种细胞中 ,转染效率也不同。经PEG表面修饰的纳米粒仍保持纳米粒的体外转染活性。连接半乳糖基牛血清白蛋白的纳米粒转染活性却反而略有下降。结论 壳聚糖纳米粒能将基因递送到细胞内 ,并且报告基因能在细胞内表达。因此 ,可以用作基因递送的载体系统 ,值得进一步研究。经PEG表面修饰和冷冻干燥处理 ,保持生物活性 ,为基因药物制剂化的可能性提供了证据。对于靶向配基的选择 ,宜继续进行筛选。

Aim To test transfection activity of DNA-chitosan nanoparticles and the change of the transfection activity after the DNA-chitosan nanoparticles were modified.Methods The DNA-chitosan nanoparticles were prepared by complex coacervation.The morphology of the particles were observed under transmission electronic microscopy(TEM).Transfection activity of DNA-chitosan nanoparticles was determined by gene transfection experiment in vitro.We test different dose of gene which is transferred and different time after the transfection to determine the best chance of the transfection.We test the change of the transfection activity,after the particles were modified with PEG and BSA-Lac.Results The morphology of the nanoparticles have no observed change after they are modified.The gene transfection experiment in vitro suggests that the DNA-chitosan nanoparticles can transfect HEK293 and HepG2 celles,and the gene can express in these celles.After the particles were modified with PEG,their transfection activity was still remained.It is important to find that in these two cell lines the transfection activity of PEGlate nanoparticles is improved.Conclusion The chitosan nanoparticles can delivery the gene into cells and the gene can express,so the chitosan nanoparticles be worth to study as gene medicine carriers.