温敏性N-异丙基丙烯酰胺—丙烯胺共聚物及其胍基衍生物非病毒基因载体

发布时间：2018-01-10 08:41

本文关键词：温敏性N-异丙基丙烯酰胺—丙烯胺共聚物及其胍基衍生物非病毒基因载体　出处：《天津大学》2006年硕士论文　论文类型：学位论文

【摘要】： 本工作采用自由基聚合制备了N-异丙基丙烯酰胺(NIPAAm)与丙烯胺(AAm)的无规共聚物(PNI-AAM),进一步将共聚物胍基化改性,生成胍基衍生物(PNI-AAM-G),并将其用于非病毒转基因载体。主要内容包括以下四部分: (1)以1H NMR及13C NMR确认了PNI-AAM与PNI-AAM-G两种共聚物的生成,元素分析结果表明产物具有较高的纯度。浊度法检测了共聚物溶液的LCST,表明随着AAM含量以及胍基化程度的增加,LCST不同程度的升高,最小LCST约为38℃。 (2)研究共聚物与DNA的相互作用情况,并考察共聚物结构和温度对其的影响。变温电泳和变温紫外结果表明,共聚物载体能有效缩合质粒DNA形成复合物,温度升高对于LCST相对较低的1-1系列聚合物的复合效果影响较为明显,各载体复合DNA的能力为:PNI-AAM 1-3 PNI-AAM-G 1-3 PNI-AAM 1-1 PNI-AAM-G 1-1。粒度检测和TEM观测考察了复合物的尺寸和形态,结果显示复合物基本为均匀的纳米粒子,粒径与载体复合能力及复合比成反向关系。 (3)以C2C12为宿主细胞,尝试了四种共聚物载体介导含Luciferase报告基因质粒的转染,通过测定luciferase的活性确定转染效率。结果表明,PNI-AAM-G 1-3共聚物载体可将pGL3质粒成功转入C2C12细胞内,总体上的转染率比裸DNA高100～400倍,比PEI高1～5倍,较脂质体低1～2个数量级,同时表现了良好的耐血清能力。若能降低此载体的LCST,使其在体温附近发生相转变,将有望通过调解温度进一步提高转染率。
[Abstract]:The free radical polymerization of N- isopropyl acrylamide was prepared (NIPAAm) and acrylamide (AAm) copolymer (PNI-AAM), the modified copolymer guanidination, generating guanidine derivatives (PNI-AAM-G), which are used for non viral vectors. The main contents include the following four parts:
(1) to 1H NMR and 13C NMR confirmed the formation of PNI-AAM and PNI-AAM-G two kinds of copolymers, the elemental analysis results showed that the product has high purity. The turbidity assay showed that with LCST copolymer solution, the content of AAM and increase the guanidination degree, LCST increased in different degree, the minimum LCST is about 38 degrees Celsius.
(2) the interaction of copolymer and DNA, and the effect of temperature on the copolymer structure and the temperature and ultraviolet. Electrophoresis results showed that copolymer carrier can effectively condensation of plasmid DNA complex formation, effect of temperature effect of composite 1-1 series polymer for LCST relatively low is obvious, ability of the carrier the compound DNA is: PNI-AAM 1-3 PNI-AAM-G 1-3 PNI-AAM 1-1 PNI-AAM-G 1-1. particle size analyzer and TEM at investigated the size and morphology of composites, the results showed that the composite material is basically uniform nanoparticles, particle size and carrier composite and composite ability than a reverse relationship.
(3) with C2C12 as the host cell, try the four kinds of copolymer carrier mediated plasmid with Luciferase gene transfection, the transfection efficiency was determined by measuring the activity of luciferase. The results showed that PNI-AAM-G 1-3 copolymer could successfully transfer pGL3 plasmid into C2C12 cells transfected with the total body rate of 100 ~ 400 higher than that of naked DNA times higher than PEI 1 to 5 times, compared with the liposome as low as 1 to 2 level, while a good serum resistance. If you can reduce the carrier of LCST, the phase transition temperature in the near, is expected to further improve the temperature through the mediation of transfection rate.

【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R318.08;R346

【共引文献】