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目的构建一种以4臂聚乙二醇(4arm PEG)为核心的串联小分子聚乙烯亚胺(PE I)的聚合物,并考察其在肿瘤基因治疗中的应用。方法应用N,N’-羰基二咪唑(N,N-’C arbony ld iim idazo le,CD I)和3-(2-吡啶二巯基)丙酸N-羟基琥珀酰亚胺酯(N-Succ in im idy l-3-(2-pyridy ld ith io)]prop ionate,SPDP)作为化学连接试剂,合成4arm PEG-PE I2000以及4arm PEG-PE I2000-M C 11,1H-NM R进行化学表征,凝胶电泳阻滞实验观察聚合物对质粒DNA的浓缩,M TT法检测聚合物的毒性,H epG 2细胞体外转基因实验确定4arm PEG-PE I2000和4arm PEG-PE I2000-M C 11的转基因能力以及M C 11的作用。结果成功合成4arm PEG-PE I2000和4arm PEG-PE I2000-M C 11,4arm PEG-PE I2000在N/P比为3时能够成功浓缩质粒DNA,毒性低,N/P比为30的时候,在H epG 2细胞中的转基因效率是PE I2000的5倍,连接上配体M C 11后,其转基因效率可以增加1倍,游离M C 11可以有效抑制4armPEG-PE I2000-M C 11的转基因效率。结论4arm PEG-PE I2000具有低毒、高转基因效率,连接配体M C 11后,可成为一个实用的转基因载体。
Objective To construct a polymer of tandem polyethyleneimine (PE I) with 4arm PEG as its core and investigate its application in tumor gene therapy. The method utilizes N,N’-carbonyldiimidazole (CDN) and 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (N-Succ) Inim idy l-3-(2-pyridy ld ith io)]prop ionate (SPDP) was chemically characterized as a chemical ligation reagent, synthesized 4arm PEG-PE I2000 and 4arm PEG-PE I2000-MC 11, 1H-NM R, Gel electrophoresis blocking experiment was used to observe the concentration of polymer on plasmid DNA, MTT was used to detect the toxicity of the polymer, and H epG 2 cells in vitro transgenic experiment confirmed the transgenic ability of 4arm PEG-PE I2000 and 4arm PEG-PE I2000-MC 11. The role of MC 11 Results The 4arm PEG-PE I2000 and 4arm PEG-PE I2000-MC 11 were successfully synthesized, and 4arm PEG-PE I2000 could successfully concentrate plasmid DNA at a N/P ratio of 3, with low toxicity and a N/P ratio of 30. The transgene efficiency in H epG 2 cells was 5 times that of PE I2000. After the ligand MC 11 was ligated, the transgene efficiency could be doubled. Free MC 11 can effectively inhibit the transgenic efficiency of 4armPEG-PE I2000-MC 11. Conclusion 4arm PEG-PE I2000 has low toxicity and high transgene efficiency. After connecting ligand M C 11, it can become a practical transgenic vector.