壳聚糖纳米粒基因载体的制备与体外转染实验研究.docVIP

壳聚糖纳米粒基因载体的制备和体外转染实验研究 （作者:___________单位: ___________邮编: ___________） 作者：王红梅，肖玉洁，韩正祥，高向阳3，裴冬生，杜秀平 【摘要】 目的 制备基因载体壳聚糖纳米粒，研究其结构特征及其体外对细胞的转染活性。方法 以复凝聚法制备壳聚糖-pDNA纳米粒;电镜测定其形态、粒径;凝胶电泳阻滞实验观察壳聚糖和pDNA的结合力;紫外可见分光光度计测定其负载力、负载率;体外转染入人胚肾细胞293T，荧光显微镜观察转染效率，CCK8法评价细胞毒性。结果 壳聚糖-pGFP 纳米粒多呈球形，直径为(132±48)nm;凝胶电泳阻滞实验示pGFP被完全包裹在纳米粒内;其负载力(LC)为(48.2±2.0)%，负载率(LE)为100%;体外转染实验证明壳聚糖-pGFP纳米粒能介导pGFP转染293T细胞并在细胞中表达绿色荧光蛋白;壳聚糖-pDNA抑制率(26.08±4.28)%。结论 采用复凝聚法制备的壳聚糖-pDNA纳米粒可有效转染至细胞内，但有一定的细胞毒性。 【关键词】 壳聚糖;纳米粒;基因载体;转染 　　Abstract:Objective To prepare chitosan nanoparticles as gene vectors so as to investigate their structural characteristics and cell-gene transfection efficiency in vitro.Methods The chitosan-pDNA nanoparticles were prepared by a complex coacervation method and their morphology as well as the particle diameter were observed under the electronic microscope. The binding of pDNA was evaluated by agarose gel electrophoresis analysis; the loading capacity and loading efficiency were determined with UV/Vis spectrophotometer. The transfection experiments were performed with human embryonic kidney 293T cell line in vitro. The transfection efficiency was measured under fluorescent microscope. The cytotoxicity was observed with Cell Counting Kit-8 (CCK-8).Results The chitosan-pDNA nanoparticles were mainly spherical, with an average diameter of (132?8)nm. The agarose gel electrophoresis analysis confirmed the DNA was wholly encapsulated in the nanoparticles. The loading capacity was (48.2?.0)% and loading efficiency was 100%. Transfection in vitro proved that chitosan-pDNA was efficient in 293T cells and the expression of green fluorescent proteins was observed under fluorescent microscope. The inhibition percentage of nanoparticles was (26.08?.28)%.Conclusions The chitosan-pDNA nanoparticles can be effectively transfected into cells. A certain degree of cytotoxicity was observed by CCK8, though. 　　Key words： chitosan; nanoparticles; gene vector; transfection 　　基因治疗为近年研究热点，高效低毒的载体为基因治疗