壳聚糖纳米粒作为肠三叶因子基因传递载体的实验研究

Experimental Study of Chitosan as Carrier of Intestinal Trefoil Factor Gene

目的构建理想的人肠三叶因子(human intestinal trefoil factor,hITF)基因传递系统。方法利用复凝法制备载基因壳聚糖纳米粒,用透射电镜观察纳米粒的形态及大小,以Zeta电位仪检测不同N/P比及不同pH对制备的载基因纳米粒Zeta电位的影响,琼脂糖凝胶电泳分析纳米粒对基因的包载能力,高速离心法测定纳米粒的包封率和载药量,分析纳米粒中基因的体外释放过程,最后检测纳米粒在HEK293细胞中的转染效率。结果利用复凝法制备了不同N/P比的壳聚糖纳米粒,透射电镜显示粒径小于1 000 nm,粒径呈窄分布;Zeta电位与N/P比呈正相关,而与pH值呈负相关;凝胶阻滞实验说明壳聚糖在N/P比大于1时可以完全包裹质粒;包封率随着N/P比的增加而增加,载药量则随着N/P比的增加而减小;体外释放实验显示,纳米粒释放速度先快后慢,并且随着N/P比的增加而逐渐减慢;壳聚糖纳米粒对HEK293细胞的转染率超过50%。结论成功构建了理想的hITF基因传递系统,为壳聚糖介导hITF基因治疗的应用打下基础。

OBJECTIVE To construct an optimal human intestinal trefoil factor （hITF） gene delivery system. METHODS Chitosan nanoparticles containing hITF gene were prepared by a complex eoacervation technique, and their size and morphology were assessed using transmission electron microscopy. Zeta potentials were determined using a Zetasizer Nano. Gel retarding ability was assayed by gel eleetrophoresis. In addition, the DNA loading efficiency and loading capacity were evaluated and the in vitro release of DNA from the chitosan nanoparticles was analyzed by high speed centrifugation. The gene transfer capability was assessed in HEK293 cells. RESULTS The chitosan nanoparticles were successfully prepared by a complex coacervation technique with sizes less than 1 000 nm and positive Zeta potentials. Release profiles of DNA were dependent on N/P ratios. The loading efficiency of chitosan nanoparticles increased in proportion to N/P ratio, while the loading capacity of nanoparticles was lower with decreasing N/P ratio. The transfection efficiencies of chitosan nanopaIticles were above 50%. CONCLUSION An optimal hlTF gene delivery system is successfully constructed. This research lays foundation for the future application of hITF gene therapy.