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。
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 ±48 ）nm. The agarose gel eleetrophoresis analysis confirmed the DNA was wholly encapsulated in the nanoparticles. The loading capacity was （48.2 ± 2.0）% and loading efficiency was 100%. Transfeetion in vitro proved that ehitosan - 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 ±4.28 ） %. Conclusions The ehitosan - pDNA nanopartieles can be effectively transfeeted into cells. A certain degree of cytotoxicity was observed by CCK8, though.
出处
《徐州医学院学报》
CAS
2010年第3期144-147,共4页
Acta Academiae Medicinae Xuzhou
基金
江苏省高校自然科学基金(08KJD320007)
徐州医学院院长专项人才科研基金(07KJZ01)
关键词
壳聚糖
纳米粒
基因载体
转染
chitosan
nanopartieles
gene vector
transfection
