壳聚糖-DNA纳米粒对肿瘤细胞的转染效率

背景非病毒载体具有低毒,低免疫反应,靶向性和易于组装等优点已成为目前研究基因转染的热点.目的评价壳聚糖-DNA纳米粒对肿瘤细胞的转染效率和细胞毒性.设计观察对比实验.单位华中科技大学同济医学院环境医学研究所.材料Zeta电位/粒度分析仪;荧光分光光度计;Hoechst 33258;PLPS-3'EGFP质粒;肺癌细胞A549和人肝癌细胞HepG2.方法实验于2004-12/2005-12在华中科技大学同济医学院环境医学研究所完成.用绿色荧光蛋白基因做报告基因,复凝聚方法制备壳聚糖绿色荧光蛋白质粒纳米粒.②采用扫描电镜观察制备的纳米粒形态;Zeta电位/粒度分析仪测量纳米粒的粒径和表面电位;酶保护试验检测纳米粒的抗DNA酶降解性能.③体外转染人肝癌细胞Hep G2和肺癌细胞A549.分别在24,48和72 h后在倒置荧光显微镜下观察细胞转染情况.④纳米粒细胞毒性分析采用四甲基偶氮唑盐试验.主要观察指标①纳米粒的包封率与理化特征.②纳米粒的细胞转染效率.③纳米粒的细胞毒性.结果①纳米粒的包封率与理化特征壳聚糖纳米粒的核酸包封率为91.7%,纳米粒多呈球形,平均粒径149 nm,表面电位+20.5 mV.壳聚糖纳米粒能保护DNA不受DNA酶Ⅰ降解.②纳米粒的细胞转染效率48 h后转染率达到高峰,A549转染率为95%;而HepG2只有10%左右.③纳米粒的细胞毒性纳米粒和壳聚糖溶液均能抑制HepG2和A549生长,壳聚糖溶液对细胞生长的抑制作用强于纳米粒.结论壳聚糖质粒纳米粒能转染HepG2和A549两种肿瘤细胞,且对两种肿瘤细胞的生长有抑制作用,提示壳聚糖纳米粒能作为DNA的载体,用于肿瘤细胞的转染.

Transfection efficiency of chitosan-DNA nanoparticles on tumor cells

BACKGROUND: At present, non-viral vectors have become a hotspot in gene transfection research because of their strong points of lower toxicity,low immunologic reaction, target orientation, and easy assembly.OBJECTIVE: To evaluate the role of chitosan-DNA nanoparticles in transfection efficiency and cellular toxicity of tumor cells.DESIGN: Observational control trial. SETTING: Institute of Environmental Medical Sciences, Tongji Medical College of Huazhong University of Science and Technology.MATERIALS: Zeta potential/ particle size analyzer; spectrofluorometer;Hoechst 33258; PLPS-3'EGFP plasmid; lung cancer cell A549 and human hepatocarcinoma cell line HepG2.METHODS: This experiment was conducted in the Institute of Environmental Medical Sciences, Tongji Medical College of Huazhong University of Science and Technology, from December 2004 to December 2005. The green fluorescent protein gene was used as report gene; chitosan green fluorescent protein plasmid nanoparticles were prepared with re-coherence gy of prepared nanoparticles; Zeta potential/ particle size analyzer was used to measure the diameter and superficial potential of the nanoparticles;enzyme-protection test was used to measure anti DNA enzyme degradation and lung cancer cell A549 were transfected in vitro. Transfection of the cells was observed under the inverted fluorescence microscope after 24, 48nanoparticles.characteristics: Nucleic acid encapsulation efficiency of chitosan nanoparticles was 91.7%, and the nanoparticles presented globular shape with the mean diameter of 149nm and superficial potential of +20.5 mV.Transfection rate of nanoparticles: It reached the peak 48 hours later; the transfection rate of A549 was 95% while that of HepG2 was only about chitosan could inhibit the growth of HepG2 and A549, and the inhibitory effect of chitosan on cellular growth was stronger than that of the nanoparticles.CONCLUSION: Nanoparticles of chitosan plasmid can transfect HepG2and A549, two kinds of tumor cells, and have inhibitory effects on their growth, suggesting that nanoparticles, as the carrier of DNA, can be used in the transfection of tumor cells.