载基因壳聚糖纳米粒的制备及免疫增强作用的初步研究

摘 要 目的: 制备壳聚糖载基因纳米粒，并对其体外转染效率及其在小鼠体内的免疫增强效果进行初步研究。方法: 以本课题组构建的口蹄疫DNA疫苗为模型药物，采用复凝聚法制备纳米粒；用透射电镜观察形态；用纳米粒度分析仪测定粒径、多分散度和zeta电位；凝胶阻滞分析测定基因在纳米粒中的位置；用体外基因转染实验评价纳米粒的转染活性。用载基因壳聚糖纳米粒免疫雌性Balb/c小鼠，检测免疫小鼠的细胞免疫和体液免疫水平。结果: 所制备的载基因纳米粒形态规则、大多成球形，平均粒径约为150nm,多分散度＜0.26,zeta电位约为21mV；凝胶分析结果表明质粒DNA与壳聚糖分子间可以通过电性结合作用而完全结合，基因几乎全部被包裹在纳米粒内部；体外基因转染实验表明壳聚糖作为一种新型的非病毒基因递送载体能够高效传递DNA进入BHK-21细胞，基因能够在该细胞中高效表达；小鼠免疫实验表明纳米粒不仅能诱导机体产生较高的细胞免疫水平，而且体液免疫水平也显著提高。结论: 壳聚糖纳米粒能将基因递送到细胞内并且能够表达，小鼠免疫实验显示其具有良好的免疫增强效果。

Preparation and immunological enhancement of chitosan nanoparticles carrying gene

Abstracts Objective: To prepare chitosan nanoparticles carrying gene，study its transfection efficiency and immunological enhancement in mice. Methods: The nanoparticles was based on the foot and mouth disease DNA vaccine which designed by our laboratory,it was prepared by complex coacervation , and its morphology was observed by transmission electronic microscopy (TEM) . Particle size , polydispersity and zeta potential were determined by nanoparticle size analyser. The molecular localization of pDNA in the chitosan nanoparticles was determined by gel retardation assay. Transfection efficiency of pDNA-chitosan nanoparticles was determined by gene transfection experiment in vitro. Balb/c mice were immunized by pDNA- chitosan nanoparticles to test both humoral and cellular immune responses. Results: The morphology of the nanoparticles were mostly spherical. The average particle size was 150nm, and the polydispersity of particle size＜0.26,the zeta potential of the nanoprticles was 21 mV. The results of gel retardation showed chitosan could completely combine the pDNA by electronic combination and pDNA was mostly encapsulated within the nanoparticles.The gene transfection experiment in vitro showed that the pDNA-chitosan nanoparticles can transfect BHK celles , and the gene can express in these celles. Immunity test proved that the nanoparticles not only induced high cellular immunogenecity, but also induced a good humoral immunogenecity in mice. Conclusion: chitosan nanoparticles can deliver gene into cell and express in these celles. Immunity test proved that it has favourable immunological enhancement.