CAJ | 학술논문

背景:裸质粒DNA在基因治疗中因带负电荷,易被体内核酸酶降解,故无法实现有效转染,壳聚糖是自然界存在的可降解性阳离子多聚糖,能有效防止DNA被核酸酶降解,提高转染效率.目的:采用复凝聚法制备包裹pcDNA3.1(-)/MAGE-3-HSP70壳聚糖纳米粒,观察其相关特性.设计、时间及地点:对比观察实验,于2009-02/08在国家卫生部纳米生物技术重点实验室完成.材料:pCDNA3.1(-)/MAGE-3-HSP70由国家卫生部纳米生物技术重点实验室构建,壳聚糖(批号060306,脱乙酰度>90.0%,黏度<100 mPa·s)由上海伯奧生物科技有限公司提供,B16细胞由中南大学肿瘤研究所惠赠.方法:采用复凝聚法制备包裹pcDNA3.1(-)/MAGE-3-HSP70壳聚糖纳米粒,将壳聚糖基因纳米粒转染B16细胞,利用反转录-聚合酶链反应检测体外转染效果;应用噻唑蓝评价壳聚糖基因纳米粒子的体外细胞毒性.主要观察指标:激光粒度仪测定壳聚糖基因纳米粒径、Zeta电位;紫外分光光度计检测包封率;凝胶阻滞实验观察壳聚糖和质粒DNA的聚合;DNase I的保护试验分析壳聚糖基因纳米粒抗核酸酶降解能力.结果:壳聚糖基因纳米粒的平均粒径约为223 nm,Zeta电位为16 mV;DNA包封率为92.3%,B16细胞转染实验显示其效率与Lipofectamine 2000相近,而其毒性远低于Lipofectamine 2000.结论:壳聚糖纳米粒子可高效装载质粒DNA转染B16细胞,而且对细胞基本无毒.
배경:라질립DNA재기인치료중인대부전하,역피체내핵산매강해,고무법실현유효전염,각취당시자연계존재적가강해성양리자다취당,능유효방지DNA피핵산매강해,제고전염효솔.목적:채용복응취법제비포과pcDNA3.1(-)/MAGE-3-HSP70각취당납미립,관찰기상관특성.설계、시간급지점:대비관찰실험,우2009-02/08재국가위생부납미생물기술중점실험실완성.재료:pCDNA3.1(-)/MAGE-3-HSP70유국가위생부납미생물기술중점실험실구건,각취당(비호060306,탈을선도>90.0%,점도<100 mPa·s)유상해백오생물과기유한공사제공,B16세포유중남대학종류연구소혜증.방법:채용복응취법제비포과pcDNA3.1(-)/MAGE-3-HSP70각취당납미립,장각취당기인납미립전염B16세포,이용반전록-취합매련반응검측체외전염효과;응용새서람평개각취당기인납미입자적체외세포독성.주요관찰지표:격광립도의측정각취당기인납미립경、Zeta전위;자외분광광도계검측포봉솔;응효조체실험관찰각취당화질립DNA적취합;DNase I적보호시험분석각취당기인납미립항핵산매강해능력.결과:각취당기인납미립적평균립경약위223 nm,Zeta전위위16 mV;DNA포봉솔위92.3%,B16세포전염실험현시기효솔여Lipofectamine 2000상근,이기독성원저우Lipofectamine 2000.결론:각취당납미입자가고효장재질립DNA전염B16세포,이차대세포기본무독.
BACKGROUND: Naked plasmid DNA cannot transfect cells effectively because of negative charge and the degradation of nuclease. Chitosan is a biodegradable natural cationic polysaccharide. It can provide effective protection against DNases and enhance transfection efficiency.OBJECTIVE: To prepare chitosan nanoparticles loaded with pcDNA3.1(-)/MAGE-3-HSP70 by complex coacervation method and to research its characteristics.DESIGN, TIME AND SETTING: A controlled experiment was performed at the Key Laboratory of Nano-biotechnology, National Ministry of Public Health of China from February to August 2009.MATERIALS: pcDNA3.1 (-)/MAGE-3-HSP70 was constructed at the Key Laboratory of Nano-biotechnology, National Ministry of Public Health of China. Chitosan, deacetylation degree > 90.0%, viscosity < 100 cps, batch number 060306, was obtained from Shanghai Bio Life Science & Technology Co., Ltd. B16 cells were presented by the Institute of Oncology, Central South University. METHODS: Chitosan nanoparticles loaded with pcDNA3.1(-)/MAGE-3-HSP70 were prepared by complex coacervation method. Then the naparticles were transfected into B16 cells, and the level of MAGE-3-HSP70 mRNA was tested using reverse transcription-polymerase chain reaction (RT-PCR) technology. The in vitro cytotoxicity of the nanoparticles was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay.MAIN OUTCOME MEASURES: Particle diameter and Zeta potential were determined by ZetaSizer 1000HSA. Efficiency of the encapsulation was measured with a spectrophotometer. The combination manner was observed by gel retardation test. The ability to protect plasmid DNA from Dnase I degradation was evaluated by DNase Ⅰ protection test. RESULTS: The mean diameter of chitosan plasmid DNA nanoparticles was 223 nm, its zeta potential was 16 mV. The encapsulation efficiency of DNA was 92.3%. The transfection efficiency of chitosan plasmid DNA nanoparticles by B16 cells was about equivalent to that of the Lipofectamine 2000 reagent. Chitosan plasmid DNA nanoparticles were much less cytotoxlc when compared with Lipofectamine 2000-pDNA complexes.CONCLUSION: Chitosan plasmid DNA nanoparticle were nontoxic to cultured cells and plasmid DNA can be efficiently transferred into B16 cells by chitosan nanoparticles.