基于聚氨基酸分子的高聚物靶向输送载体的研究
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摘要
基因输送技术在几十年发展中,取得了令人瞩目的成果,但还存在一些问题急需解决。目前最重要的是构建安全有效的能将基因输送到特定位置的基因输送载体,其中非病毒载体由于安全性、易于修饰等优点受到广泛关注。非病毒载体除骨架外,为了达到某些特定的目的需要进行表面修饰以增加体外、体内的输送效果,如在载体表面修饰配体可以增加基因输送的靶向性,提高在特定组织或细胞中的基因表达;在载体表面修饰亲水性聚合物如聚乙二醇(PEG)等可以延长基因输送系统在体内的循环时间。
本文对修饰后的载体结构进行了深入研究之后,发现现有的方法制备出的非病毒基因输送载体系统存在难以定量控制、表征修饰过程和产物的问题。因而提出采用寡聚谷氨酸将功能分子间接地修饰到复合粒子的表面,有利于对复合粒子表面修饰的结构和数量更有效地控制,并且在细胞试验中取得了较好的转染效果的方法。
首先本文简要介绍了基因治疗方法、回顾了基因治疗载体的研究进展,并对各种基因治疗载体做了介绍。着重对非病毒载体作了系统地阐述包括聚赖氨酸(PLL)、脂质体、聚乙二亚胺(PEI)、阳离子多肽等的目前状况、研究进展。还介绍了阳离子聚合物载体的主要组成成分和表征方法以及阐述了本论文的意义。
本论文工作首先应用毛细管电泳技术研究了靶向分子——表皮生长因子(EGF)和表面保护分子——PEG化学接枝的聚赖氨酸载体的结构和分布。EGF、PEG与PLL通过交联剂连接后,常常获得的是具有多种结构的混合物。我们将DNA和PLL或PEG-g-PLL以一定的比例混合形成复合粒子,复合粒子的组成也相当复杂。
为了对表面修饰分子以及复合粒子有更好的质量控制,我们设计了一个20个谷氨酸的聚合物,PEG或EGF修饰后,再通过聚谷氨酸上游离羧基的负电性以静电引力吸附到DNA/PLL复合粒子表面,表征了不同比例的PLL、DNA与PLG、EGF-g-PLG或PEG-g-PLG混合形成的复合物的物理化学性质包括粒径、表面电位、荧光替代和表面形貌等。我们提出了一种方便、快捷的高聚物载体修饰方法,同时对这些复合粒子的生物学性质进行了表征,包括在有血清和无血清的培养基中的细胞转染效率。获得了一些有意义的结果,对以后的工作具有一定的指导作用。
本论文不仅为目前的载体系统提供一个可供借鉴的质量控制方法,而且展示了一种可以广泛使用的阳离子高聚物载体系统组装方法,为以后使用的新型阳离子高聚物载体遇到类似的问题也能够从本论文的研究方法得到借鉴。
During the development of gene delivery techniques in recent decades, there are so many problem needing to be dissolved, although a lot of fruits have been made. Now it is the most important that construct the gene delivery vector which can safely and efficiently deliver gene to the specific tissue or cell. However the amount of modification on the surface of vector or complexes through the existing preparation methods was difficultly determined. In our paper, the function molecule was indirectly modified on the surface of complexes by poly-l-glutamic acid as a bridge, which can well control the amount of modification on the surface of complexes and obtained preferable transfection efficiency.
Firstly, the development of gene therapy vector, the brief introduction of various vector, the main components in and characterization of polycation vector and the meaning about our paper was reviewed in our paper.
Then capillary electrophoresis was used to characterize the reaction products between PLL and EGF or PEG. And the complexes formed with PLL, PEG-g-PLL and DNA at certain charge ratio was characterized by capillary electrophoresis.
The PLG modified with EGF or PEG through the sole end amino of PLG, which was characterized by capillary electrophoresis, was electrostaticly adsorbed to the surface of DNA/PLL complexes, because there is too close charge to mass ratio in the reaction products between PLL and EGF or PEG to be separated by capillary electrophoresis. Then PLL, DNA, EGF-g-PLG and/or PEG-g-PLG at various charge ratio was formed into complexes, and characterization about the physicochemical properties of these complexes such as size, zeta potential, EtBr displacement and morphology was done. Also the biological property of these complexes was characterized, which include the cell transfection efficiency in medium with or without serum.
In sum, we systemically studied a vector with convenient and fast modification, which was done the characterization of physicochemical properties and cell transfection experiments in which the preferable results was obtained. All these results can direct future work in assembly system.
本文对修饰后的载体结构进行了深入研究之后,发现现有的方法制备出的非病毒基因输送载体系统存在难以定量控制、表征修饰过程和产物的问题。因而提出采用寡聚谷氨酸将功能分子间接地修饰到复合粒子的表面,有利于对复合粒子表面修饰的结构和数量更有效地控制,并且在细胞试验中取得了较好的转染效果的方法。
首先本文简要介绍了基因治疗方法、回顾了基因治疗载体的研究进展,并对各种基因治疗载体做了介绍。着重对非病毒载体作了系统地阐述包括聚赖氨酸(PLL)、脂质体、聚乙二亚胺(PEI)、阳离子多肽等的目前状况、研究进展。还介绍了阳离子聚合物载体的主要组成成分和表征方法以及阐述了本论文的意义。
本论文工作首先应用毛细管电泳技术研究了靶向分子——表皮生长因子(EGF)和表面保护分子——PEG化学接枝的聚赖氨酸载体的结构和分布。EGF、PEG与PLL通过交联剂连接后,常常获得的是具有多种结构的混合物。我们将DNA和PLL或PEG-g-PLL以一定的比例混合形成复合粒子,复合粒子的组成也相当复杂。
为了对表面修饰分子以及复合粒子有更好的质量控制,我们设计了一个20个谷氨酸的聚合物,PEG或EGF修饰后,再通过聚谷氨酸上游离羧基的负电性以静电引力吸附到DNA/PLL复合粒子表面,表征了不同比例的PLL、DNA与PLG、EGF-g-PLG或PEG-g-PLG混合形成的复合物的物理化学性质包括粒径、表面电位、荧光替代和表面形貌等。我们提出了一种方便、快捷的高聚物载体修饰方法,同时对这些复合粒子的生物学性质进行了表征,包括在有血清和无血清的培养基中的细胞转染效率。获得了一些有意义的结果,对以后的工作具有一定的指导作用。
本论文不仅为目前的载体系统提供一个可供借鉴的质量控制方法,而且展示了一种可以广泛使用的阳离子高聚物载体系统组装方法,为以后使用的新型阳离子高聚物载体遇到类似的问题也能够从本论文的研究方法得到借鉴。
During the development of gene delivery techniques in recent decades, there are so many problem needing to be dissolved, although a lot of fruits have been made. Now it is the most important that construct the gene delivery vector which can safely and efficiently deliver gene to the specific tissue or cell. However the amount of modification on the surface of vector or complexes through the existing preparation methods was difficultly determined. In our paper, the function molecule was indirectly modified on the surface of complexes by poly-l-glutamic acid as a bridge, which can well control the amount of modification on the surface of complexes and obtained preferable transfection efficiency.
Firstly, the development of gene therapy vector, the brief introduction of various vector, the main components in and characterization of polycation vector and the meaning about our paper was reviewed in our paper.
Then capillary electrophoresis was used to characterize the reaction products between PLL and EGF or PEG. And the complexes formed with PLL, PEG-g-PLL and DNA at certain charge ratio was characterized by capillary electrophoresis.
The PLG modified with EGF or PEG through the sole end amino of PLG, which was characterized by capillary electrophoresis, was electrostaticly adsorbed to the surface of DNA/PLL complexes, because there is too close charge to mass ratio in the reaction products between PLL and EGF or PEG to be separated by capillary electrophoresis. Then PLL, DNA, EGF-g-PLG and/or PEG-g-PLG at various charge ratio was formed into complexes, and characterization about the physicochemical properties of these complexes such as size, zeta potential, EtBr displacement and morphology was done. Also the biological property of these complexes was characterized, which include the cell transfection efficiency in medium with or without serum.
In sum, we systemically studied a vector with convenient and fast modification, which was done the characterization of physicochemical properties and cell transfection experiments in which the preferable results was obtained. All these results can direct future work in assembly system.
引文
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