羧基对壳聚糖—硬脂酸嫁接物介导的基因传递的影响
发布时间：2018-11-24 14:51
【摘要】：本研究为了进一步增加壳聚糖硬脂酸的转染效率,分别从化学嫁接和物理混合两种方式引入羧基,以研究促进CSO-SA基因转染效率的方法,并探讨其机理。 通过碳二亚胺法,将小分子阴离子-顺乌头酸化学嫁接到CSO-SA上得到CA-CSO-SA。氢核磁共振确证了嫁接物的化学组成。经TNBS法测得CSO-SA和CA-CSO-SA的实际氨基取代度为4.03%和6.31%,临界胶束浓度80.3μg/L和78.4μg/L。微粒粒度与表面电位分析仪测得1.0mg/mL的CSO-SA和CA-CSO-SA胶束的数均粒径为55.3±7.6nm和60.0±7.1nm,电位为38.3±2.8mV和30.8±1.0mV。透射电子显微镜结果显示这两种胶束呈不规则的球形,粒径较小,分布均一。 分别制备了CSO-SA/DNA, CA-CSO-SA/DNA复合物,虽然在顺乌头酸修饰后,CA-CSO-SA与DNA密接略有下降但是依然可以有效的保护DNA免受酶降解的能力,且形成的复合物具有良好的血清稳定性。CA-CSO-SA/DNA复合物在HEK293细胞介导的转染效率达到37%,与CSO-SA/DNA相比,转染效率增加一倍,优于阳性对照lipofectamineTM2000。虽然顺乌头酸修饰降低了细胞摄取的速率,但是在24h时细胞摄取总量是没有差异的。CA-CSO-SA/DNA在HEK293细胞中,主要通过网格蛋白介导的内吞作用,以及小窝蛋白介导的胞吞作用和巨胞饮作用。CSO-SA/DNA复合物容易陷在内溶酶体中,但CA-CSO-SA/DNA能够更有效的从内溶酶体中逃逸出来并分布在细胞质中。顺乌头酸修饰提高了CSO-SA的生物安全性,且在转染剂量下对细胞基本不呈现毒性。 将聚谷氨酸PGA分别与DNA混合后,再与CSO-SA嫁接物胶束混合,制备不同N/P/C比CSO-SA/DNA/PGA三元复合物。随着PGA增加,三元复合物起始粒径变化不大,而在N/P/C比为10/1/6时粒径显著性增加,而多分散系数以及电位都随着PGA的加入而出现明显的下降趋势。以绿色荧光蛋白质粒(pEGFP-C1)为报告基因,流式细胞仪测定细胞转染百分率的结果表明CSO-SA/DNA/PGA三元复合物的基因转染效率随着PGA先增加后降低；在N/P/C=10/1/4时转染效率为27.4%,是CSO-SA/DNA二元复合物的两倍,且接近阳性LipofectamineTM2000对照,以虫荧光素酶质粒为报告基因,化学发光仪测定其发光值得结果表明CSO-SA/DNA/PGA三元复合物在N/P/C=10/1/4虫荧光素酶的表达最高,达到5.32*105RLU/mg Protein,约是二元复合物表达的10倍。共聚焦观察细胞内过程和Bafilomycin Al抑制实验结果显示CSO-SA/DNA/PGA复合物具有更高的溶酶体逃逸的效率归结于三元复合物质子缓冲能力的增加,从而获得了更高效的基因转染效率。 研究结果表明化学嫁接或者物理混合适量的负电荷都是有利于CSO-SA介导的基因转染,且引入的羧基对于促进内溶酶体的逃逸起了重要作用。
[Abstract]:In order to further increase the transfection efficiency of chitosan stearic acid, carboxyl groups were introduced from two ways of chemical grafting and physical mixing in order to study the method of promoting the transfection efficiency of CSO-SA gene and its mechanism. Chemical grafting of small anions and cisaconitric acid onto CSO-SA to obtain CA-CSO-SA. by carbodiimide method The chemical composition of the graft was confirmed by HNMR. The actual degree of amino substitution for CSO-SA and CA-CSO-SA was 4.03% and 6.31, and the critical micelle concentration was 80.3 渭 g / L and 78.4 渭 g / L by TNBS method. The average particle size of CSO-SA and CA-CSO-SA micelles in 1.0mg/mL were 55.3 卤7.6nm and 60.0 卤7.1 nm, the potential were 38.3 卤2.8mV and 30.8 卤1.0 MV, respectively. The results of transmission electron microscope show that the two micelles are irregular spherical, smaller in size and uniform in distribution. The CSO-SA/DNA, CA-CSO-SA/DNA complexes were prepared separately. Although the close binding of CA-CSO-SA to DNA decreased slightly after modification of aconitate, it could still effectively protect DNA from enzymatic degradation. The transfection efficiency of CA-CSO-SA/DNA complex reached 37% in HEK293 cells. Compared with CSO-SA/DNA, the transfection efficiency of CA-CSO-SA/DNA complex was twice that of CSO-SA/DNA, which was better than that of positive control lipofectamineTM2000.. Although the rate of uptake was decreased by aconitonic acid modification, there was no difference in total uptake at 24 h. CA-CSO-SA/DNA in HEK293 cells mainly mediated endocytosis by grid protein. CSO-SA/DNA complex is easy to sink into the inner lysosome, but CA-CSO-SA/DNA can escape from the inner lysosome and distribute in the cytoplasm more effectively. The modification of aconitonic acid improved the biosafety of CSO-SA and showed no toxicity to the cells at the transfection dose. Poly (glutamate) PGA was mixed with DNA and then mixed with CSO-SA grafted micelle to prepare CSO-SA/DNA/PGA ternary complexes with different N/P/C ratios. With the increase of PGA, the initial particle size of the ternary complex does not change much, but the particle size increases significantly when the N/P/C ratio is 10-1-6, while the polydispersity coefficient and potential decrease obviously with the addition of PGA. Using green fluorescent protein (pEGFP-C1) as reporter gene, the transfection rate of CSO-SA/DNA/PGA ternary complex was determined by flow cytometry. The transfection efficiency of CSO-SA/DNA/PGA ternary complex increased firstly with PGA and then decreased. The transfection efficiency at N/P/C=10/1/4 was 27.4%, which was twice as high as that of the binary complex of CSO-SA/DNA, and was close to the positive LipofectamineTM2000 control. The plasmids of luciferase were used as reporter genes. The results of chemiluminescence assay showed that the expression of CSO-SA/DNA/PGA ternary complex in luciferase of N/P/C=10/1/4 worm was the highest, and the expression of 5.32*105RLU/mg Protein, was about 10 times as high as that of binary complex. The results of confocal observation of intracellular processes and Bafilomycin Al inhibition experiments showed that the higher efficiency of lysosomal escape of CSO-SA/DNA/PGA complex was attributed to the increase of proton buffer capacity of ternary complexes. Thus, a more efficient gene transfection efficiency was obtained. The results show that chemical grafting or physical mixing with proper amount of negative charge is beneficial to CSO-SA mediated gene transfection and the introduction of carboxyl groups plays an important role in promoting the escape of internal lysosomes.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943
本文编号：2354137
[Abstract]:In order to further increase the transfection efficiency of chitosan stearic acid, carboxyl groups were introduced from two ways of chemical grafting and physical mixing in order to study the method of promoting the transfection efficiency of CSO-SA gene and its mechanism. Chemical grafting of small anions and cisaconitric acid onto CSO-SA to obtain CA-CSO-SA. by carbodiimide method The chemical composition of the graft was confirmed by HNMR. The actual degree of amino substitution for CSO-SA and CA-CSO-SA was 4.03% and 6.31, and the critical micelle concentration was 80.3 渭 g / L and 78.4 渭 g / L by TNBS method. The average particle size of CSO-SA and CA-CSO-SA micelles in 1.0mg/mL were 55.3 卤7.6nm and 60.0 卤7.1 nm, the potential were 38.3 卤2.8mV and 30.8 卤1.0 MV, respectively. The results of transmission electron microscope show that the two micelles are irregular spherical, smaller in size and uniform in distribution. The CSO-SA/DNA, CA-CSO-SA/DNA complexes were prepared separately. Although the close binding of CA-CSO-SA to DNA decreased slightly after modification of aconitate, it could still effectively protect DNA from enzymatic degradation. The transfection efficiency of CA-CSO-SA/DNA complex reached 37% in HEK293 cells. Compared with CSO-SA/DNA, the transfection efficiency of CA-CSO-SA/DNA complex was twice that of CSO-SA/DNA, which was better than that of positive control lipofectamineTM2000.. Although the rate of uptake was decreased by aconitonic acid modification, there was no difference in total uptake at 24 h. CA-CSO-SA/DNA in HEK293 cells mainly mediated endocytosis by grid protein. CSO-SA/DNA complex is easy to sink into the inner lysosome, but CA-CSO-SA/DNA can escape from the inner lysosome and distribute in the cytoplasm more effectively. The modification of aconitonic acid improved the biosafety of CSO-SA and showed no toxicity to the cells at the transfection dose. Poly (glutamate) PGA was mixed with DNA and then mixed with CSO-SA grafted micelle to prepare CSO-SA/DNA/PGA ternary complexes with different N/P/C ratios. With the increase of PGA, the initial particle size of the ternary complex does not change much, but the particle size increases significantly when the N/P/C ratio is 10-1-6, while the polydispersity coefficient and potential decrease obviously with the addition of PGA. Using green fluorescent protein (pEGFP-C1) as reporter gene, the transfection rate of CSO-SA/DNA/PGA ternary complex was determined by flow cytometry. The transfection efficiency of CSO-SA/DNA/PGA ternary complex increased firstly with PGA and then decreased. The transfection efficiency at N/P/C=10/1/4 was 27.4%, which was twice as high as that of the binary complex of CSO-SA/DNA, and was close to the positive LipofectamineTM2000 control. The plasmids of luciferase were used as reporter genes. The results of chemiluminescence assay showed that the expression of CSO-SA/DNA/PGA ternary complex in luciferase of N/P/C=10/1/4 worm was the highest, and the expression of 5.32*105RLU/mg Protein, was about 10 times as high as that of binary complex. The results of confocal observation of intracellular processes and Bafilomycin Al inhibition experiments showed that the higher efficiency of lysosomal escape of CSO-SA/DNA/PGA complex was attributed to the increase of proton buffer capacity of ternary complexes. Thus, a more efficient gene transfection efficiency was obtained. The results show that chemical grafting or physical mixing with proper amount of negative charge is beneficial to CSO-SA mediated gene transfection and the introduction of carboxyl groups plays an important role in promoting the escape of internal lysosomes.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943
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相关期刊论文 前3条
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