胆固醇在PAMAM介导的基因传递过程中的作用

发布时间：2018-06-10 07:16

本文选题：聚酰胺-胺 + 脂质体　；参考：《天津大学》2008年硕士论文

【摘要】： 聚酰胺-胺(PAMAM,polyamidoamine)以其独有的分子结构和物理特性,能够通过静电疏水等作用与DNA紧密结合,并将其缩聚,保护DNA不被降解,是近年来广泛使用的非病毒基因治疗载体。但是相比病毒载体,PAMAM的转染效率仍然很低,这是由于它在转染过程中存在多个屏障。对于PAMAM/DNA复合物与细胞结合并进入细胞的具体机制的理解,克服PAMAM转染过程的细胞膜屏障,将有助于我们找到提高PAMAM转染效率方法。本文采用卵磷脂脂质体模拟细胞膜骨架,并在其中加入胆固醇单组分,通过等温微量量热法,Zeta电势和透射电子显微镜,对PAMAM/DNA复合物与脂质体的相互作用进行考察,探索PAMAM/DNA复合物与脂质体膜的作用机制,以及胆固醇在其中所起到的作用。热力学研究表示,对于含有和不含有胆固醇的脂质体,PAMAM/DNA复合物主要通过静电作用与其表面结合,反应由焓驱动,胆固醇的加入将使结合在表面的PAMAM/DNA复合物能够嵌入脂双层中,从而引起焓变、熵变的增大,甚至出现聚集的吸热过程。PAMAM/DNA复合物的电荷比大于1时,脂质体的负电势随着PAMAM/DNA复合物的加入而减小,说明两者通过静电作用结合,胆固醇在静电结合的过程中没有作用。通过电镜观察,对于不加胆固醇的脂质体,PAMAM/DNA复合物结合在脂质体表面,可以看到两球结合的状态,对于加入胆固醇的脂质体,PAMAM/DNA复合物进入脂质体内部,脂质体粒径增大,仍为球状。各种检测均表明PAMAM/DNA复合物通过静电作用结合在表面后,通过胆固醇的作用进入细胞膜。
[Abstract]:Polyamidoamine (PAMAM) is a widely used non-viral gene therapy vector in recent years, because of its unique molecular structure and physical properties, it can be tightly combined with DNA by electrostatic hydrophobicity and condensed to protect DNA from degradation. However, the transfection efficiency of PAMAM is still very low compared with that of virus vector pama, which is due to the existence of multiple barriers during the transfection process. Understanding the specific mechanism of PAMAM / DNA complex binding to cells and entering cells, overcoming the membrane barrier during PamAm transfection will help us to find a way to improve the transfection efficiency of PamAm. In this paper, lecithin liposome was used to simulate the membrane skeleton. Cholesterol monocomponent was added to it. The interaction between PamAm / DNA complex and liposome was investigated by means of isothermal microcalorimetry Zeta potential and transmission electron microscope, and the mechanism of the interaction between PamAm / DNA complex and liposome membrane was explored. Thermodynamic studies show that for liposomes containing and without cholesterol, the pama / DNA complex is mainly bound to its surface by electrostatic interaction, and the reaction is driven by enthalpy. When cholesterol is added, the PAMAM- / DNA complex that binds to the surface can be embedded in the lipid bilayer, resulting in enthalpy changes, entropy changes, and even the aggregation of endothermic processes. The charge ratio of PAMAM- / DNA complexes is greater than 1. The negative potential of liposome decreases with the addition of PamAm / DNA complex, indicating that cholesterol has no effect on electrostatic binding. The observation of electron microscope showed that when the liposome without cholesterol was bound to the surface of the liposome, it could be seen that the two spheres were bound. For the liposome containing cholesterol, the pama / DNA complex entered into the liposome, and the diameter of the liposome increased. Still spherical. The results showed that the PAMAM / DNA complex was bound to the surface by electrostatic interaction and entered the cell membrane by cholesterol.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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