PDMAEMA及其共聚物的基因传递性能研究

发布时间：2018-04-18 11:05

本文选题：基因传递 + 聚甲基丙烯酸-N　；参考：《天津科技大学》2017年硕士论文

【摘要】：基因治疗是指将基因片段(如DNA)导入靶细胞的一种治疗疾病的方式,从而让外源基因纠正或修复基因缺陷、关闭或抑制异常表达的基因,从而达到治愈疾病的目的。基因治疗作为一种新型的治疗癌症的方法,在临床方面已经取得了重大进展。聚甲基丙烯酸-N, N' -二甲氨基乙酯(PDMAEMA)作为一种研究细胞转染效率与聚合物结构之间关系的模型,在基因载体研究领域内受到广泛的关注,而且它具有优良的转基因性能,有望在未来成为基因传递领域的突破点。本文首先采用原子转移自由基聚合法(ATRP)在水溶液中制备了一系列不同聚合度的PDMAEMA均聚物和PEG-b-PDMAEMA嵌段共聚物。通过红外光谱、核磁共振谱等测试方法确定均聚物和共聚物在聚合物结构、相对分子量等方面均符合预期。紫外可见分光光度计的光透射率-温度测试表明PDMAEMA和PEG-b-PDMAEMA均具有温度敏感性,并且研究了影响聚合物低临界溶解温度(LCST)的多种因素,从而验证聚合物的LCST是否会影响其作为基因载体的基因传递性能。本项目以人结肠癌细胞(HCT-116)作为研究对象,采用编码有Flag蛋白标签的pCMV-3×Flag-LTA4H大肠杆菌进行质粒DNA的扩培、提取,利用带正电的聚合物能与带负电的DNA静电相互作用自组装形成纳米微球的原理,将合成的PDMAEMA均聚物和PEG-b-PDMAEMA嵌段共聚物应用于基因传递方面,以商品化的Lipofectamine 2000 Reagent(Life)转染剂为参照,研究所制备聚合物的基因传递性能,从细胞转染效率和细胞毒性等多方面进行了评价。结果表明,PDMAEMA均聚物有较高的体外转染效率,同时也具有了一定的细胞毒性。PEG化可以改善基因载体的应用,尽管PEG化后会降低转染效率,但它能够降低细胞毒性。更为重要的是,PEG可以作为一种连接体,为进一步提高载体性能提供广阔的发展空间。
[Abstract]:Gene therapy is a way to treat diseases by introducing gene fragments (such as DNA) into target cells, so that exogenous genes can correct or repair gene defects, turn off or inhibit abnormal expression of genes, and achieve the goal of curing diseases.Gene therapy, as a new method of cancer treatment, has made great progress in clinical practice.As a model to study the relationship between cell transfection efficiency and polymer structure, poly (methacrylate), N '-dimethylaminoethyl ester (PDMAEMA), has attracted extensive attention in the field of gene vector research, and it has excellent transgenic performance.It is expected to become a breakthrough point in the field of gene transmission in the future.In this paper, a series of PDMAEMA homopolymers and PEG-b-PDMAEMA block copolymers with different degrees of polymerization were prepared by atom transfer radical polymerization (ATRP) in aqueous solution.The structure and relative molecular weight of homopolymers and copolymers were determined by IR, NMR and other methods.The phototransmittance and temperature measurements of UV-Vis spectrophotometer show that both PDMAEMA and PEG-b-PDMAEMA are temperature-sensitive, and many factors affecting the low critical solution temperature of polymer are studied.To verify whether the polymer's LCST will affect its gene transfer performance as a gene vector.The human colon cancer cell line HCT-116) was used as the research object. The plasmid DNA was amplified and extracted by pCMV-3 脳 Flag-LTA4H E. coli with Flag protein tag.Based on the principle that polymer with positive charge can interact with negatively charged DNA to form nano-microspheres, the synthesized PDMAEMA homopolymers and PEG-b-PDMAEMA block copolymers were used in gene transfer, as compared with commercial Lipofectamine 2000 ReagentLife-based transcoloring agent.The gene transfer properties of the prepared polymers were evaluated in terms of cell transfection efficiency and cytotoxicity.The results showed that PDMAEMA homopolymer had a high transfection efficiency in vitro and a certain cytotoxicity. PEGylation could improve the application of the gene vector. Although the transfection efficiency was decreased after PEG, it could reduce the cytotoxicity.More importantly, PEG can be used as a kind of connectors, which provides a broad development space for further improving the performance of the carrier.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R450

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