【摘要】 目的制备巯基化壳聚糖,构建巯基化壳聚糖-质粒DNA(pDNA)纳米粒,并研究该纳米粒对HeLa细胞的体外转染活性。方法1-(3-二甲胺基丙基)-3-乙基碳二亚胺盐酸盐(EDAC)催化巯基乙酸与壳聚糖反应生成巯基化壳聚糖,傅里叶变换红外光谱仪测量产物的红外光谱,5-5’-二硫代-双-硝基苯甲酸(DTNB)法检测产物的巯基含量;以pcDNA 3.1(+)-EGFP为报告基因,巯基化壳聚糖为载体,用复凝聚法制得巯基化壳聚糖-pcDNA3.1(+)-EGFP纳米粒,Zeta电位和粒度分析仪检测该纳米粒的表面电位和粒径。纳米粒经DNaseI处理、再在肝素作用下解聚,用琼脂糖凝胶电泳检测质粒的完整性。评价巯基化壳聚糖-pcDNA 3.1(+)-EGFP纳米粒对HeLa细胞的体外转染活性,MTT法测定该纳米粒对HeLa细胞的毒性。结果红外光谱图显示壳聚糖在EDAC的作用下被巯基化。DTNB法显示1g巯基化壳聚糖的巯基含量为(202.85±3.05)μmoL(n=6)。Zeta电位及粒度分析仪的结果表明巯基化壳聚糖-pcDNA 3.1(+)-EGFP纳米粒的平均粒径为288.7nm,Zeta电位为+(25.2±2.1)mV。DNaseⅠ保护实验证明该纳米粒能有效抑制DNaseⅠ对内部pDNA的降解。体外转染实验表明巯基化壳聚糖-pcDNA 3.1(+)-EGFP纳米粒能有效转染HeLa细胞系,且对HeLa细胞生长无抑制作用。结论该制备工艺生产的巯基化壳聚糖-pcDNA 3.1(+)-EGFP纳米粒能将质粒DNA导入HeLa细胞,使报告基因有效地表达。所以巯基化壳聚糖是基因转运和基因治疗中有应用潜力的非病毒类载体。更多还原

【Abstract】 【Objectives】To study the preparation of thiolated chitosan and thiolated chitosan-plasmid DNA (pDNA) nanoparticles, and to investigate transfection activities of thiolated chitosan-based nanoparticles to Hela cells in vitro.【Methods】Thiolated chitosan was prepared in the synthesis reaction of chitosan and thioglycolic acid by the catalysis of 1-thyl-3-(3-dimethylaminopropyl)-carbodoomide hydrochloride (EDAC). Infrared spectrum of product was measured by Fourier transform infrared detector,and the degree of thiol groups modification of thiolated chitosan was determined by 5, 5’-dithiobis (2-nitrobenzoiol acid) (DTNB) assay. Nanoparticles were synthesized through complex coacervation thiolated chitosan vector with pcDNA 3.1 (+)-EGFP, the Latter acting as the reporter gene. The diameter and surface charge of the nanoparticles were determined by Zetasize and grainsize analyzer. The nanoparticles were treated by DNase I, then depolymerized by heparin, and integrity of plasmid gene was measured by gel electrophoresis. The transfection activities of the thiolated chitosan-pcDNA 3.1 (+)-EGFP nanoparticles to HeLa cells were assessed in vitro. The cytotoxity of the nanoparticles to HeLa cells was examined with the MTT assay. 【Results】Infrared spectrum showed that chitosan is thiolatized by the catalysis of EDAC. DTNB assay exhibit that 1 gram of thiolated chitosan processed (202.85±3.05) μmol thiol groups (n=6). Grainsize and Zetasize analyzer showed thiolated chitosan-pcDNA 3.1(+)-EGFP nanoparticles were 288.7 nm in average size and + (25.2±2.1) mV of zeta electric potential. The encapsulated pDNA protection against DNase I degradation was confirmed by DNase I protection test. The nanoparticles were shown to transfect HeLa cells effectively in transfection experiment in vitro and showed no cytotoxicity to HeLa cells. 【Conclusions】Reporter genes were effectively expressed by the HeLa cells when transfected by thiolated chitosan-pcDNA 3.1 (+)-EGFP nonaparticles constructed by the technology. So thiolated chitosan may serve as a promising nonviral gene vector for gene transfer and gene therapy.更多还原