Wan Yi-Yuan, Zhang Xing, He You-Jian, Jiang Wen-Qi
State Key Laboratory of Oncology in Southern China, Guangzhou, Guangdong, 510060, PR China.
Ai Zheng. 2005 Nov;24(11):1408-11.
BACKGROUND & OBJECTIVE: Recently, chitosan, as a nonviral vehicle for transferring DNA molecules into the cells, has attracted much attention because of its cationic properties. This study was to investigate characteristics and transfection activity of chitosan-pDNA microparticles, and confirm the feasibility of chitosan as gene therapy vehicle, which may be employed in further in vivo study.
Plasmid DNAs were amplified in Eschericha Coli JM109 with transfection of enhanced green fluorescence protein (EGFP) gene, and isolated according to the protocol of Qiagen plasmid midi kit. Chitosan-pDNA microparticles were prepared by the coacervation method, and observed under transmission electronic microscope. In vitro releasing experiment and ultraviolet spectrophotometry were used to measure DNA loading capacity and loading efficiency. The location of pDNA in the microparticles was analyzed by gel retardation assay. Transfection efficiency of chitosan-pDNA microparticles was evaluated by detecting gene expression of EGFP in HEK293 cells after transfection.
Chitosan-pDNA microparticles were global, and the size ranged 100-200 nm, with the average of (138 +/- 43) nm. DNA loading capacity and loading efficiency of the microparticles were (46.8 +/- 9.0)% and 100%, respectively. Gel retardation assay confirmed that DNA was wholly encapsulated in the microparticles. In vitro study revealed that the release of DNA from chitosan-pDNA microparticles included two-step process, namely burst effect with a slow second phase. Most of DNA could be released at the time of 48 h. In vitro transfection results indicated that chitosan could efficiently deliver pEGFP into HEK293 cells and stably express green florescence protein.
Chitosan can efficiently transfer target DNA molecules into mammalian cells, continuously release DNA, and stably express DNA. It can be potentially employed as a gene therapy vehicle.
近年来，壳聚糖作为一种将DNA分子导入细胞的非病毒载体，因其阳离子特性而备受关注。本研究旨在探讨壳聚糖-pDNA微粒的特性和转染活性，并证实壳聚糖作为基因治疗载体的可行性，以便进一步用于体内研究。
用增强型绿色荧光蛋白（EGFP）基因转染，在大肠杆菌JM109中扩增质粒DNA，并按照Qiagen质粒中量提取试剂盒的方案进行分离。采用凝聚法制备壳聚糖-pDNA微粒，在透射电子显微镜下观察。通过体外释放实验和紫外分光光度法测定DNA包封率和包封效率。用凝胶阻滞试验分析pDNA在微粒中的定位。通过检测转染后HEK293细胞中EGFP的基因表达来评估壳聚糖-pDNA微粒的转染效率。
壳聚糖-pDNA微粒呈球形，大小在100-200nm之间，平均为（138±43）nm。微粒的DNA包封率和包封效率分别为（46.8±9.0）%和100%。凝胶阻滞试验证实DNA完全包裹在微粒中。体外研究表明，壳聚糖-pDNA微粒中DNA的释放包括两个阶段，即突发效应和随后的缓慢释放阶段。大部分DNA在48小时时释放。体外转染结果表明，壳聚糖能有效地将pEGFP导入HEK293细胞并稳定表达绿色荧光蛋白。
壳聚糖能有效地将目标DNA分子导入哺乳动物细胞，并持续释放DNA和稳定表达DNA。它有可能作为一种基因治疗载体。
