【作者】 柏金根；

【导师】 姚康德；

【作者基本信息】 天津大学 ， 材料学， 2007， 硕士

【摘要】 本工作采用均相反应法制备N-亚甲基磷酸化壳聚糖衍生物（NMPCS）,研究了其与DNA的复合能力,并将所形成的复合物用于介导体外基因转染,同时考察了载体及其复合物的细胞毒性,主要内容包括以下四个部分:（1）以红外光谱（FT-IR）分析表明改性后的壳聚糖的特征基团有显著变化;13CNMR检测进一步证明了亚甲基磷酸基团的引入,结合元素分析确定了取代度。（2）通过复凝聚的方法,制备了纳米尺寸的NMPCS/DNA聚电解质复合物。考察了共聚物与DNA的相互作用情况,复合物中DNA的构象变化和复合物的形态和粒径。圆二色谱结果表明DNA与壳聚糖作用形成纳米尺寸的复合物后仍然保持着典型的B型构象。透射电镜,原子力显微镜检测结果表明,不同N/P下的N-亚甲基磷酸化修饰的壳聚糖/DNA纳米复合物呈现较规则的球形,且在高电荷比时可形成尺寸较小的纳米尺度颗粒。（3）以人肝癌细胞HepG2和人宫颈癌细胞HeLa为宿主细胞,尝试了以NMPCS为载体介导含荧光素酶的pGL3质粒的体外转染,重点研究了pH值和N/P比对转染效率的影响。实验结果表明,载体可有效将质粒转染HeLa和HepG2细胞,获得的最佳转染效率远高于裸DNA和CS,与PEI相当。基于磷酸基团对钙离子有良好的鳌合能力制备了聚合物盐型NMPCS-Ca载体,并用于介导基因转染,其转染过程类似于传统的P-Ca载体,并认为Ca²⁺离子通道促进了转染过程。考察了16-CS和NMPCS载体协同介导基因转染研究,结果表明亲疏水相互作用影响复合物的形成和基因转染活性。（4）利用MTT实验初步研究了衍生物载体及其复合物在上述两种细胞中的毒性,结果表明在实验条件下,载体和复合物的毒性要远远小于同等浓度时PEI的毒性,而载体的毒性要稍高于对应复合物的毒性,且体现一定的浓度依赖性。更多还原

【Abstract】 In this study, N-methylene phosphonic chitosan (NMPCS), an amphiphilic macromolecule with powerful chelating ability of Ca2+ ions, was synthesized and characterized, the interactions between NMPCS and plasmid DNA, the gene transfection and delivery mediated by NMPCS/DNA complexes, and cell cytotoxcity of the derivative and their complexes were also investigated. The main work is summarized as follows:(1) N-methylene phosphonic chitosan (NMPCS) was synthesized using a one-step reaction that allowed homogeneous modifications without any sharp decrease in its properties. FT-IR, 13CNMR, and element analysis permitted the identification of the structure and the degree of substitution of NMPCS.(2) NMPCS/DNA complexes were obtained using a complex coacervation process, characterized by agarose gel electrophoresis retardation assay for their stabilities, atomic force microscopy (AFM), transmission electron microscopy (TEM) and laser size analysizer observation for morphology and size, results showed that at charge ratio 2:1 or above, DNA could be completely entrapped and spherical complexes with mean size of 80~210nm were formed, CD spectra verified that the interaction between DNA and NMPCS caused a slight perturbation on DNA bonds and DNA still remained B-conformation within the complexes.(3) Complexes were further employed to transfect HeLa and HepG2 cells, and transfection efficiency was investigated using the pGL3 luciferase reporter gene in cell culture medium with different pH values. The results indicated that the transfection efficiencies were strongly dependent on the charge ratio and pH value of culture medium, for HeLa cells, the highest efficiency obtained at ratio of 4:1 and pH 6.2 was greatly higher than that from CS/DNA complexes or naked DNA and approximate to that from PEI/DNA complexes, although HepG2 cells were less efficiently transfected. Polymer-inorganic salt type NMPCS-Ca vector showed some similarities with traditional Ca-P, giving high efficiencies probably owe to positive role of Ca2+ ion channel. Synergistic effect of 16 N-dodecylated chitosan(16-CS)on the transfection efficiency of NMPCS /DNA complexes indicated that hydrophobic and hydrophilic interactions influenced the formation of the complexes and thus the transfection process.(4) The cytotoxity of derivative alone and its complexes with plasmid DNA were determined by 3-(4, 5-dimethylthiazd-2-yl)-2, 5-diphenylt-entrazolium bromide (MTT) assay both on HepG2 and HeLa cells, the results suggested that the derivative alone and their complexes showed significantly lower toxicity than PEI and PEI/DNA complexes, respectively, and Nevertheless, both NMPCS alone and its complexes also showed a dose-dependent cytotoxicity.更多还原