叶酸修饰壳聚糖靶向转基因载体的研究

发布时间：2018-04-15 12:34

本文选题：叶酸 + 壳聚糖　；参考：《武汉工业学院》2009年硕士论文

【摘要】： 目前,壳聚糖作为一种新型非病毒基因载体,已引起越来越多的国家和研究机构的重视。叶酸—叶酸受体靶向是近年来倍受青睐的一种新型抗肿瘤机制,它是利用叶酸受体在某些肿瘤部位的过度表达而在正常组织低水平表达的特性而实现目的基因的靶向输送。因此,用叶酸对壳聚糖进行化学修饰,可提高基因转染的靶向性和转染效率。本实验利用壳聚糖氮原子上的酰化反应,缩合剂首先与叶酸生成活性中间体,进一步与壳聚糖上的氨基反应制备叶酸偶联壳聚糖。实验中制备了不同连接比例的叶酸偶联壳聚糖,采用红外光谱法验证叶酸与壳聚糖的偶联,用紫外分光光度法测定每个壳聚糖上连接叶酸的数量;选择绿色荧光蛋白基因作为报告基因,用复凝聚法制备叶酸偶联壳聚糖-DNA纳米粒和壳聚糖-DNA纳米粒,用透射电镜观察纳米微粒表面形态,通过DNA酶保护性检测实验观察叶酸偶联壳聚糖-DNA纳米粒和壳聚糖-DNA纳米粒对质粒的保护作用;利用培养的HeLa细胞进行转染,通过荧光显微镜定性观察,比较叶酸偶联壳聚糖纳米粒和壳聚糖纳米粒作为基因治疗载体的优劣。叶酸偶联壳聚糖红外光谱图表明叶酸与壳聚糖成功偶联,每个壳聚糖上连接叶酸的数量大约为3个、10个、20个、30个;实验中制备的叶酸偶联壳聚糖-DNA纳米粒溶液和壳聚糖-DNA纳米粒溶液为外观均一、稳定的透明溶液;透射电子显微镜检测结果显示,纳米粒分布均匀,大小一致,无粘合,外观呈球形或近球形,叶酸偶联壳聚糖-DNA纳米粒粒径大约为120nm左右,壳聚糖-DNA纳米粒粒径大约为140nm左右;DNA酶保护性检测实验表明壳聚糖纳米粒和叶酸偶联壳聚糖纳米粒均有良好的DNA保护作用;荧光显微镜检测结果显示,叶酸偶联壳聚糖-DNA纳米粒和壳聚糖-DNA纳米粒均能将绿色荧光蛋白质粒基因递送到细胞内并表达产生绿色荧光蛋白,但每个壳聚糖上连接10个和20个叶酸的叶酸偶联壳聚糖-DNA纳米粒转染效果更好,有更高的靶向性和转染效率。
[Abstract]:At present, chitosan as a novel non viral gene vector, has attracted more and more countries and research institutions pay much attention. Folic acid - folate receptor targeting is a new anti-tumor mechanism in recent years is popular, it is characteristic in the over expression of some kinds of tumor and normal tissue expression in the low level of use of folate receptor the realization of the target of gene transfer. Therefore, the chemical modification of chitosan with folic acid, can improve the targeting of gene transfection and transfection efficiency.
This experiment using the acylation reaction of Chitosan on nitrogen atom, condensation agent and folic acid first generation of reactive intermediates, further reacted with amino groups of the preparation of Chitosan on Preparation of folate conjugated chitosan. Weprepared different folate conjugated chitosan connection ratio, coupling by using infrared spectroscopy to prove the amount of folate with chitosan. The connection of folic acid in each chitosan was determined by UV spectrophotometry; selection of green fluorescent protein gene as a reporter gene, using complex coacervation of folate conjugated chitosan -DNA nanoparticles and chitosan -DNA nanoparticles by using transmission electron microscope observation of surface morphology of nano particles, the DNA enzyme protection assay. The protective effect of folate chitosan -DNA nanoparticles and chitosan -DNA nanoparticles on plasmid; transfection by cultured HeLa cells by fluorescence microscope, comparison of folate conjugated chitosan Nanoparticles and chitosan nanoparticles are used as gene therapy carriers.
Folate conjugated chitosan IR spectra indicate that the coupling of folate with chitosan was successful, chitosan on the number of connections each folic acid is about 3, 10, 20, 30; in the experiment of preparation of folate conjugated chitosan -DNA nanoparticles solution and chitosan -DNA nanoparticles solution for appearance are a stable, transparent solution; transmission electron microscopy showed that nanoparticles of uniform distribution, the same size, no adhesion, the appearance of a spherical or nearly spherical, folate coupled chitosan -DNA nanoparticle size is about 120nm, chitosan -DNA nanoparticle size is about 140nm; DNA enzyme detection experiments show that DNA protective protective effect of chitosan nanoparticles and folic acid conjugated chitosan nanoparticles were good; fluorescence microscopy showed that folate conjugated chitosan -DNA nanoparticles and chitosan -DNA nanoparticles can be green fluorescent protein gene delivery The green fluorescent protein was expressed in the cells, but the folate conjugated chitosan -DNA nanoparticles with 10 folate and 20 folate on each chitosan had better transfection efficiency and higher targeting and transfection efficiency.

【学位授予单位】：武汉工业学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346

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