多肽接枝壳聚糖—聚乙烯亚胺—聚乙二醇作为基因载体的制备及在神经再生中的应用研究
发布时间：2019-06-12 17:55
【摘要】：运用基因转染技术可以实现对细胞蛋白表达的调控,其中构建高效低毒的基因载体是关键。神经营养因子-3(NT-3)能促进神经干细胞(NSC)向神经元的分化和繁殖,维持神经元存活,但是其在体内不稳定,易被降解,而被转染了NT-3基因的受体细胞可不断地表达NT-3。本课题利用功能短肽修饰的阳离子聚合物构建了一类基因载体,探讨了将NT-3基因转导入受体细胞的表达及对NSC分化的影响作用。具体工作如下:(一)将壳聚糖氧化开环后与PEI接枝(CS-g-PEI),具有双官能团的PEG通过加成反应与短肽接枝(PEG-PEP),最后CS-g-PEI和PEG-PEP经酰胺反应连接形成CS-g-PEI-PEG-PEP。实验结果表明四元聚合物能成功合成,电位和粒径分布适宜,呈现为结构紧密的圆球形,能有效包裹DNA且对细胞的毒性较小,复合物进入NIH-3T3和Hela、293T细胞后报告基因能从中释放出来并被转录表达。(二)利用CS-g-PEI-PEG-PEP将NT-3基因运载至受体细胞后,运用酶联免疫吸附试验(ELISA)检测得受体细胞能有效表达NT-3,用含有转染后受体细胞培养液的分化培养基培养NSC,NSC分化成神经元的比例提高,且神经元轴突长度增加,存活率更高。
[Abstract]:Gene transfer technology can be used to regulate the expression of cell protein, in which the construction of high efficiency and low toxicity gene vector is the key. Neurotrophic factor-3 (NT-3) can promote the differentiation and reproduction of neural stem cell (NSC) into neurons and maintain the survival of neurons, but it is unstable and easy to degrade in vivo. The recipient cells infected with NT-3 gene can constantly express NT-3.. In this study, a class of gene vectors were constructed by using cationic polymers modified by functional peptides to investigate the expression of NT-3 gene into receptor cells and its effect on the differentiation of NSC. The specific work is as follows: (1) chitosan was oxidized and opened up and grafting with PEI (CS-g-PEI). PEG with bifunctional groups was Graft with short peptides (PEG-PEP) by addition reaction. Finally, CS-g-PEI and PEG-PEP were connected by amide reaction to form CS-g-PEI-PEG-PEP.. The results showed that the quaternion polymer could be synthesized successfully, the potential and particle size distribution were suitable, and it showed a compact spherical structure, which could effectively encapsulate DNA and had little toxicity to cells. The reporter gene could be released and expressed in NIH-3T3 and Hela,293T cells after the complex entered NIH-3T3 and Hela,293T cells. (2) after NT-3 gene was carried to recipient cells by CS-g-PEI-PEG-PEP, (ELISA) assay was used to detect the effective expression of NT-3, into neurons. The proportion of NSC,NSC differentiated into neurons in differentiation medium containing transferred receptor cell culture medium was increased, and the axonal length of neurons was increased and the survival rate was higher.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R450
[Abstract]:Gene transfer technology can be used to regulate the expression of cell protein, in which the construction of high efficiency and low toxicity gene vector is the key. Neurotrophic factor-3 (NT-3) can promote the differentiation and reproduction of neural stem cell (NSC) into neurons and maintain the survival of neurons, but it is unstable and easy to degrade in vivo. The recipient cells infected with NT-3 gene can constantly express NT-3.. In this study, a class of gene vectors were constructed by using cationic polymers modified by functional peptides to investigate the expression of NT-3 gene into receptor cells and its effect on the differentiation of NSC. The specific work is as follows: (1) chitosan was oxidized and opened up and grafting with PEI (CS-g-PEI). PEG with bifunctional groups was Graft with short peptides (PEG-PEP) by addition reaction. Finally, CS-g-PEI and PEG-PEP were connected by amide reaction to form CS-g-PEI-PEG-PEP.. The results showed that the quaternion polymer could be synthesized successfully, the potential and particle size distribution were suitable, and it showed a compact spherical structure, which could effectively encapsulate DNA and had little toxicity to cells. The reporter gene could be released and expressed in NIH-3T3 and Hela,293T cells after the complex entered NIH-3T3 and Hela,293T cells. (2) after NT-3 gene was carried to recipient cells by CS-g-PEI-PEG-PEP, (ELISA) assay was used to detect the effective expression of NT-3, into neurons. The proportion of NSC,NSC differentiated into neurons in differentiation medium containing transferred receptor cell culture medium was increased, and the axonal length of neurons was increased and the survival rate was higher.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R450
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5 要e，
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