【作者】 刘炯；

【导师】 郭圣荣；

【作者基本信息】 上海交通大学 ， 药剂学， 2007， 硕士

【摘要】 本文研究了硬脂酰鱼精蛋白的合成,并将其作为非病毒基因载体,考察了与质粒DNA的复合性能,复合物的体外基因转染效果。主要内容包括以下三部分:1)通过N-羟基琥珀酰亚胺(NHS)与硬脂酸形成的活泼酯,在水性反应环境中与鱼精蛋白进行酯交换反应,从而将疏水性的硬脂酰基团偶联到鱼精蛋白上,成功合成了三种硬脂酰鱼精蛋白(stearyl protamine,简称SP),分别为SP1,SP2,SP3。将SP在HCl中降解后回收硬脂酸,并用酸碱微量滴定的方法测定含量。SP中硬脂酰基团的含量(质量百分比)分别为2.5%、3.0%、5.5%。通过动态光散射(DLS)对SP以及硫酸鱼精蛋白(PS)的研究发现,SP3可以在水中自组装形粒径110nm左右的稳定聚集体,此聚集体的粒径不随SP3浓度的提高而发生变化,说明是一种热力学稳定的自组装聚集体。2)通过工程菌扩增,碱法抽提,获得编码萤火虫荧光素酶的质粒DNA pGL3-control,并以此作为后继实验中采用的DNA材料。凝胶电泳阻滞实验检验SP对DNA的复合能力,结果表明当载体/DNA质量比超过1.0时,DNA均能被三种SP完全包装起来。与未修饰的鱼精蛋白相比,随着SP中硬脂酰基团含量的升高,其对DNA的复合能力略有减弱。对鱼更多还原

【Abstract】 In this thesis, stearyl protamine as a kind of novel non-viral gene vector was synthesized and investigated on the characters of its DNA complexes and the transfection efficiency. The main work includes three parts as follows:1) Three kinds of stearyl protamine (SP) named as SP1, SP2 and SP3 were synthesized of coupling protamine with stearic acid in an aquatic solution system via the active ester formed by NHS and stearic acid. The content of stearyl group (w/w %) of SP was measured by acid-base microtitration of stearic acid obtained from the degradation of SP in HCL. SP1 contains 2.5% stearyl group, 3.0% for SP2 and 5.5% for SP3. Results of DLS measurement showed that SP3 would form stable aggregates in water with around 110 nm in average diameter. The size of SP3 aggregates remained equal when the concentration of SP3 raised up, which indicated that SP3 aggregates were self-assembled and thermodynamic stable in water.2) Plasmid pGL3-control, which encoding firefly luciferase was used as DNA material in the experiments and obtained from bioengineer bacteria. The result of gel retardation assay showed that DNA could be fully compacted by SP when carrier/DNA weight ratio above 1.0. Compared with that of natural protamine sulfate, the DNA package ability of SP slightly dropped with the increasing of stearyl group. Size measurement showed that all SP/DNA complexes were smaller than 150nm in average diameter at the evaluated weight ratios. Polyplexes of SP1/DNA and SP2/DNA have the sizes similar to that of protamine sulfate/DNA but SP3/DNA polyplexes were much larger. Since SP3 would form stable self-aggregates in water. This indicated a new pattern for SP3 to compact DNA. DNase I protection assay showed that SP/DNA complexes could protect DNA from degradation by DNase I.3) H1299-human lung cancer cell was used as the subject during the MTT and in virto transfection experiments. Results showed that the cytotoxicity of SP was low at the dosage ranged from 2.5μg/ml ~ 30μg/ml. No cytotoxicity was added with the increase of stearyl更多还原