【摘要】 背景:裸DNA在基因治疗中易被体内核酸酶降解。携载DNA疫苗的病毒类载体存在致癌和引发免疫应答等安全问题,且制备方法复杂。非病毒载体(尤其是阳离子-DNA聚合物)具有低毒,低免疫反应,能重复给药,易于大量制备等优点。壳聚糖是一种天然阳离子碱性聚多糖,具有良好的生物可降解性、生物相容性及生物安全性。目的:制备含质粒pHEV23的纳米壳聚糖,观察其表征及对DNA的保护作用。设计、时间及地点:对比观察实验,于2007-06/2008-05在浙江省医学科学院生物工程研究所完成。材料:壳聚糖,脱乙酰度88%,批号061220,由青岛利中甲壳质公司提供。质粒pHEV23为浙江省医学科学院生物工程研究所实验室提供。方法:通过脱水剂Na2SO4诱导的复凝聚沉淀法制备含质粒pHEV23的纳米壳聚糖。根据壳聚糖质量浓度1.4,0.7,0.4g/L,分别制备壳聚糖(高)-pHEV23、壳聚糖(中)-pHEV23、壳聚糖(低)-pHEV23。主要观察指标:激光纳米粒度分析仪测定壳聚糖-pHEV23粒径、Zeta电位;紫外分光光度计检测包封率;凝胶阻滞实验分析壳聚糖和pHEV23的聚合;DNaseⅠ的保护试验分析壳聚糖-pHEV23的抵抗核酸酶降解能力;释放实验评价壳聚糖-pHEV23的稳定性。结果:制备的壳聚糖-pHEV23粒径在170～470nm,其中壳聚糖(低)-pHEV23171.6nm,壳聚糖(中)-pHEV23387.0nm,壳聚糖(高)-pHEV23467.3nm。壳聚糖(低)-pHEV23Zeta电位为+22.9mV。包封率均>95%,其中壳聚糖(低)-pHEV2395.8%,壳聚糖(中)-pHEV2396.7%,壳聚糖(高)-pHEV2397.1%。凝胶阻滞分析表明,壳聚糖和pHEV23之间通过静电作用完全结合,纳米粒带正电荷。DNaseⅠ保护试验表明,壳聚糖-pHEV23能有效抵抗DNaseⅠ酶降解,对pHEV23有保护作用。稳定性试验表明,4℃保存60d,纳米粒仍能较稳定地包裹pHEV23。结论:壳聚糖-pHEV23能高效装载外源DNA,稳定性良好,并保护其免受核酸酶的降解。更多还原

【Abstract】 BACKGROUND:Naked DNA is easily degraded by nuclease in a body during gene therapy.Viral vector carrying DNA vaccine has some problems in carcinogenesis and immune response initiation,with a complicated preparation method.Non-viral vector(especially cation-DNA polymer) has low toxin and low immune response,and can be repetitively administered,and is easily prepared in a large number.Chitosan,a natural cation basic polysaccharides,has good biodegradability,biocompatibility and biological safety.OBJECTIVE:To prepare chitosan nanoparticles carrying pHEV23 as gene carriers,and to study its characteristics and ability to protect DNA.DESIGN,TIME AND SETTING:The controlled experiment was performed at the Institute of Bioengineering,Zhejiang Academy of Medical Science from June 2007 to May 2008.MATERIALS:Chitosan,degree of deacetylation of 88%,batch number 061220,was obtained from Lizhong Chitin Co.,Ltd.Plasmid pHEV23 was obtained from Laboratory of Institute of Bioengineering,Zhejiang Academy of Medical Science.METHODS:Chitosan nanoparticles(CS)-pHEV23 were prepared using a complex coacervation process induced by Na2SO4.According to mass concentration of chitosan(1.4,0.7,0.4 g/L),chitosan(high)-pHEV23,chitosan(middle)-pHEV23 and chitosan(low)-pHEV23 were prepared.MAIN OUTCOME MEASURES:Chitosan-pHEV23 particle diameter and Zeta potential were determined using laser nanoparticle analyzer.Efficiency of the encapsulation was measured with a spectrophotometer.The combination manner of CS with pHEV23 was analyzed by gel retardation test.The ability to protect pHEV23 from nuclease degradation were evaluated using DNase Ⅰ protection test.Chitosan-pHEV23 stability was assessed by release test.RESULTS:The particle diameter of the nanoparticles were from 170 nm to 470 nm(Chitosan(low)-pHEV23 171.6 nm,Chitosan(middle)-pHEV23 387.0 nm,Chitosan(high)-pHEV23 467.3 nm).Zeta potential(Chitosan(low)-pHEV23) +22.9 mV;the efficiency of the encapsulation were over 95%(Chitosan(low)-pHEV23 95.8%,Chitosan(middle)-pHEV23 96.7%,Chitosan(high)-pHEV23 97.1%).The gel retardation assay showed that chitosan could completely combine with pHEV23 by electrostatic effect,with positive charge.The DNase Ⅰ protection test confirmed that pHEV23 could be protected from DNaseⅠ degradation.The stability test confirmed that at 4 ℃ the pHEV23 could be parcelled well for 60 days.CONCLUSION:Chitosan-pHEV23 can effectively protect DNA from DNaseⅠ degradation,with a good stability.更多还原