糖皮质激素—聚乙烯亚胺作为新型基因给药系统的研究

发布时间：2017-12-30 22:32

本文关键词：糖皮质激素—聚乙烯亚胺作为新型基因给药系统的研究　出处：《浙江大学》2010年硕士论文　论文类型：学位论文

【摘要】： 本研究旨在利用糖皮质激素(GC)与细胞质中糖皮质激素受体(GR)特异性结合后转移入细胞核的能力,合成一种具有细胞核靶向能力,低毒、高效的新型阳离子非病毒基因给药载体,并考察了其体内体外的转染效果。采用了效价(potency)和LogP评价GC结构与转染结果的关系。利用Traut's试剂将5种糖皮质激素:倍他米松(BEX),地塞米松(DEX),甲基泼尼松龙(MPL)、泼尼松龙(PNL)和氢化可的松(HC)接枝到PEI 1800,得到了5种GC-PEIs,取代度都在1左右。通过琼脂糖凝胶电泳,缓冲能力擦拭,DLS粒径测定和Zeta电位测定实验检测了GC-PEIs/pDNA的理化性质,五种GC-PEIs与pDNA的复合物理化性质相似。然而,体外HEK 293和HepG2两种细胞系上的细胞毒性和转染效率测定结果都证实了GC的修饰显著提高了PEI 1800的转染效率,但是五种GC之间有明显差异。针对GC的结构与转染效果的关系考察,采用了分子模拟对接程序考察了5种GC和GR的结合情况,并比较了用效价和LogP值来评价转染效果的可行性。同时,采用激光共聚焦显微镜对载体在细胞内进行定位,发现BET-PEI基本实现了细胞核靶向的目的。小鼠体内肝门静脉注射BET-PEI包载pEGFP-N1复合物,倒置荧光显微成像和流式细胞术FCM测定都再一次证明了BET-PEI能显著提高转染效率,并超过PEI 25k的水平。
[Abstract]:The purpose of this study is to use Glucocorticoid (GC) and glucocorticoid receptor in the cytoplasm (GR) specific binding capacity after the transfer into the nucleus, with synthesis of a nuclear targeting ability, low toxicity, a new cationic efficient non viral gene delivery vectors, and investigated its in vitro and in vivo transfection effect. The titer of the relationship between LogP (potency) and evaluation of GC structure and transfection results.
Using Traut's reagent 5 glucocorticoid betamethasone (BEX), dexamethasone (DEX), methyl (MPL), P Nixonn Ron P Nixonn Ron (PNL) and hydrocortisone (HC) grafted to PEI 1800, we got 5 kinds of GC-PEIs, the degree of substitution are 1.
By agarose gel electrophoresis, buffer capacity wipe, DLS particle size and Zeta potential were measured to detect the physicochemical properties of GC-PEIs/pDNA composite, physical and chemical properties of five kinds of GC-PEIs is similar with pDNA. However, in vitro determination of HEK 293 and HepG2 two cell lines on the cytotoxicity and transfection efficiency are confirmed by the results of the modification of GC significantly improve the transfection efficiency of PEI 1800, but there is significant difference between the five kinds of GC. The influences of relationship between the structure and the transfection efficiency of GC, using the molecular docking program was investigated with 5 kinds of GC and GR, and the feasibility to evaluate the transfection efficiency value with titer and LogP.
At the same time, the location within the cell carrier by laser scanning confocal microscope, BET-PEI found the basic realization of the purpose of nuclear targeting. Mice hepatic portal vein injection of BET-PEI loaded pEGFP-N1 composite inverted fluorescence microscopy and flow cytometry determination of FCM have once again proved that BET-PEI can significantly improve the transfection efficiency, and more than the PEI 25K level.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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