双亲基因载体的构筑及在增强内皮细胞转染效率方面的研究

发布时间：2018-02-24 04:04

本文关键词： 内皮细胞转染效率两亲性基因载体靶向识别自组装　出处：《天津大学》2015年博士论文　论文类型：学位论文

【摘要】：目前,心脑血管疾病手术治疗存在的主要问题是移植物中新生内膜增生以及再狭窄等,这些都严重危及患者生命。鉴于内皮细胞的一些天然优势,植入材料表面快速内皮化是材料在体内移植成功的关键。基因治疗和材料表面的功能化修饰是实现人工血管植入材料内皮化的两种有效方法。本论文系统地综述了基因治疗和多肽修饰的生物材料在促进内皮化方面取得的成果。由于内皮细胞固有的转染效率低下,使植入材料在体内的内皮化效果大受影响。因此,有效提高内皮细胞的转染效率对于促进人工血管材料的内皮化有着最为直接的意义。鉴于阳离子基因载体的分子量和靶向活性多肽对基因的传递有重要影响,本论文首先探讨了阳离子基因载体的分子量对内皮细胞转染效率的影响,进而又设计了多肽修饰的新型基因载体,并用于内皮细胞的体外转染研究。第二章.为了考察阳离子基因载体的分子量对内皮细胞转染效率的影响,本章以聚甲基丙烯酸羟乙酯(PHEMA)为载体主链,通过引发L-丙交酯(LA)开环聚合,得到PHEMA-PLA嵌段共聚物。不同分子量的亲水链聚乙二醇单甲醚(PEG)和聚乙烯亚胺(PEI)通过酯化和酰胺化反应连接到羧基化的PHEMA-PLA嵌段共聚物上,制备得到四种不同分子量的阳离子基因载体:PHEMA-PLA-PEG 2kDa-PEI 1.8 kDa、PHEMA-PLA-PEG 5 kDa-PEI 1.8 kDa、PHEMA-PLA-PEG 2kDa-PEI 10 kDa和PHEMA-PLA-PEG 5 kDa-PEI 10 kDa。进而通过溶液自组装的方法分别构建得到四种对应的纳米粒:G2I1.8、G5I1.8、G2I10和G5I10。采用动态光散射技术考察了这四种纳米粒载体及其与pEGFP-ZNF580基因形成的复合物的流体力学直径和表面zeta电位的大小。并选取EA.hy926为模型内皮细胞,考察这四种基因载体及其复合物对内皮细胞的生物学毒性及转染活性的影响。结果表明适当地提高阳离子基因载体的分子量和在载体中引入PEG有助于提高载体复合物在内皮细胞中的转染效率。第三章.本章制备了一种REDV(Arg-Glu-Asp-Val)多肽修饰的基因载体,用于负载pEGFP-ZNF580基因,考察了靶向活性多肽修饰的载体复合物在内皮细胞中的转染效率。通过开环聚合和接枝聚合的方法制备得到聚乙二醇单甲醚-聚(丙交酯-co-己内酯)-聚乙烯亚胺(mPEG-P(LA-co-CL)-PEI)两亲性嵌段共聚物。通过CREDVW多肽与mPEG-P(LA-co-CL)-PEI的共价键合反应,制备得到REDV多肽修饰的基因载体mPEG-P(LA-co-CL)-PEI-REDV。并通过自组装的方法制备得到聚合物mPEG-P(LA-co-CL)-PEI-REDV和mPEG-P(LA-co-CL)-PEI对应的纳米粒载体REDV-NP和NP。通过凝胶电泳的方法考察了两种载体纳米粒对pEGFP-ZNF580基因的包覆能力。以EA.hy926为模型内皮细胞,借助MTT实验考察了载体基因复合物对内皮细胞生物毒性的影响,并考察了载体复合物在内皮细胞中的转染效率。实验结果表明,靶向活性多肽REDV修饰的基因载体对pEGFP-ZNF580基因包覆能力强,对内皮细胞生物相容性较好,体外转染效率比PEI 25 kDa复合物高,且能有效促进内皮细胞的迁移。第四章.在本章研究中,为了制备多位点连接REDV多肽修饰的基因载体,考察其在内皮细胞中递送基因的能力,我们以PHEMA主链为大分子引发剂,采用开环聚合的方法将己内酯引入到PHEMA的多条臂肢中,进而通过酯化反应和酰胺化反应将PEG和小分子量的PEI与羧基化后的PCL链端连接,制备得到两亲性的梳型基因载体PHEMA-PCL-PEG-PEI。借助一种异双官能团连接剂,将REDV活性多肽引入到PEI链端,制备得到REDV多肽功能化的两亲性基因载体(PHEMA-PCL-PEG-PEI-REDV)。再通过自组装的方法构筑了这种聚合物纳米粒,用于对pEGFP-ZNF580基因的包载。通过动态光散射技术和凝胶成像电泳分析的方法考察了靶向载体对pEGFP-ZNF580基因包覆能力的大小。以EA.hy926细胞为模型细胞,考察了靶向基因载体复合物对内皮细胞的生物毒性及在内皮细胞中的转染效果,并对细胞中的蛋白表达含量进行测定。结果表明该REDV多肽修饰的双亲梳型基因载体可有效包载pEGFP-ZNF580基因,且不会对内皮细胞造成严重的细胞毒性,在内皮细胞中转染效率好,相应的蛋白表达含量高。实验结果表明阳离子基因载体的分子量对内皮细胞的转染效率有着很大的影响,且生物活性多肽修饰的载体及基因复合物可有效提高内皮细胞的体外转染效率。本文的研究工作对提高生物材料的内皮化有着潜在的指导意义,有望应用于心脑血管疾病的介入治疗中。
[Abstract]:At present, the main problems of surgical treatment of cardiovascular and cerebrovascular diseases is the graft neointimal hyperplasia and restenosis, which seriously endanger the lives of patients. In view of some natural advantages of endothelial cells, implant materials quick endothelialization of materials is the key to success in the transplantation in vivo. Functional modification of the gene therapy and the surface of the material is two effective methods of artificial vascular endothelial implants. This thesis systematically summarizes the gene therapy and the peptide modified biological material made in promoting endothelialisation achievements. Due to the low transfection efficiency of endothelial cell intrinsic, make implant materials in vivo endothelialization effect greatly. Therefore, effectively improve the transfection of endothelial cells to promote the efficiency of endothelialization of artificial blood vessel materials have the most direct significance. In view of the molecular weight and cationic gene vector targeting activity Peptides have important influence on gene transfer, this paper discusses the influence of molecular weight of cationic gene vector transfection efficiency, and then design a new gene carrier peptide modified, and used for transfection of endothelial cells in vitro. In the second chapter. In order to investigate the effect of molecular weight of cationic gene vector transfection the efficiency of this chapter to poly 2-hydroxyethyl methacrylate (PHEMA) as the carrier backbone, by triggering L- lactide ring opening polymerization (LA), PHEMA-PLA block copolymer. The hydrophilic polyethylene glycol with different molecular weight (PEG) and polyethyleneimine (PEI) through esterification and amidation of carboxyl attached to the PHEMA-PLA block copolymer, prepared four kinds of different molecular weight cationic gene vector: PHEMA-PLA-PEG 2kDa-PEI 1.8 kDa, PHEMA-PLA-PEG 5 kDa-PEI 1.8 kDa, PHEMA-PLA-PEG 2kDa-PEI 10 KDa and PHEMA-PLA-PEG 5 kDa-PEI 10 kDa. respectively and then construct four corresponding nanoparticles by solution self-assembly: G2I1.8, G5I1.8, fluid mechanics, composite diameter and surface zeta potential of G2I10 and G5I10. on these four kinds of nanoparticles and pEGFP-ZNF580 gene formed by dynamic light scattering technology and select EA.hy926 size. As a model of endothelial cells, biological toxicity and transfection activity to examine these four gene vector and its compounds on endothelial cells. The results show that properly increasing the molecular weight and cationic gene vector PEG was introduced into the carrier is helpful to improve the carrier complex in endothelial cells transfection efficiency. In the third chapter, a kind of REDV. Were prepared in this chapter (Arg-Glu-Asp-Val) gene carrier peptide modified, used to load pEGFP-ZNF580 gene, investigated the carrier complex targeting peptide modified The efficiency of transfection in endothelial cells. Methods through ring opening polymerization and graft polymerization to prepare polyethylene glycol monomethyl ether poly (-co- lactide caprolactone) - polyethyleneimine (mPEG-P (LA-co-CL) -PEI) two amphiphilic copolymers by CREDVW peptide and mPEG-P (LA-co-CL) -PEI covalent bond reaction. Preparation of mPEG-P peptide modified REDV gene vector (LA-co-CL) and -PEI-REDV. by self-assembly method prepared by polymer mPEG-P (LA-co-CL) -PEI-REDV and mPEG-P (LA-co-CL) -PEI corresponding to REDV-NP and NP. nanoparticles were investigated with two carrier nanoparticles on pEGFP-ZNF580 gene by gel electrophoresis method with EA.hy926 as a model. With the help of MTT endothelial cells, the effects of the gene complex on endothelial cell toxicity, and the effects of the carrier complex in endothelial cells in transfection efficiency. The experimental results Show that the gene vector targeting peptide modified REDV of pEGFP-ZNF580 gene with strong ability of endothelial cells have good biocompatibility, in vitro transfection efficiency than the PEI 25 kDa complex, and can effectively promote the migration of endothelial cells. In the fourth chapter. In this chapter the research, in order to gene carrier preparation of multilocus REDV connection peptide modified gene delivery, investigate its ability in endothelial cells, we used PHEMA as the backbone of macroinitiator, using the method of ring opening polymerization of multiple limbs caprolactone into PHEMA, then the PEG and PEI of small molecular weight and carboxylated PCL after the chain end connected by esterification the reaction and amidation reaction, preparation of two amphiphilic comb type gene vector PHEMA-PCL-PEG-PEI. with the help of a hetero bifunctional linker, REDV active polypeptide into the PEI chain end, preparation of two amphiphilic functionalized REDV polypeptide gene carrier Body (PHEMA-PCL-PEG-PEI-REDV). Through the self-assembly method to build the polymer nanoparticles for the encapsulation of pEGFP-ZNF580 gene. Methods by dynamic light scattering and gel electrophoresis analysis examines the imaging ability of pEGFP-ZNF580 gene targeting vector coated size. Using EA.hy926 cells as model cells, effects of targeting gene carrier complex biological toxicity of endothelial cells and in endothelial cells and the cell transfection effect, the protein expression levels were determined. The results showed that the REDV peptide modified amphiphilic comb gene carrier can be effectively loaded pEGFP-ZNF580 gene, and will not cause severe cytotoxicity to endothelial cells, endothelial cell transfection efficiency well, the corresponding protein content is high. The experimental results show that the transfection efficiency of cationic gene vector on endothelial cells has a great influence The carrier, and biological activity of peptide modified and gene complexes can effectively improve transfection efficiency in vitro endothelial cells. This research work has potential significance for improving endothelialization of biomaterials, interventional therapy is expected to be applied to cardiovascular and cerebrovascular diseases.

【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08;R54

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