PAMAM树状化合物作为呼吸道合胞病毒基因疫苗递送载体的研究

发布时间：2018-01-29 18:14

本文关键词： DNA疫苗载体 PAMAM树状化合物细胞毒性免疫　出处：《北京交通大学》2010年硕士论文　论文类型：学位论文

【摘要】： DNA疫苗能刺激机体产生细胞免疫与体液免疫,其制造成本低廉,易于储存及运输。但是,裸DNA疫苗在体内传输过程中,很容易被酶水解,不能引起足够的免疫原性,很难达到好的免疫保护效果。DNA递送载体能够保护DNA分子不被酶水解。本课题选取聚酰胺-胺(PAMAM)树状化合物作为基因疫苗递送载体,通过细胞毒性实验及细胞凋亡实验研究了PAMAM G4及PAMAM G5在多种细胞系中的毒性,在此基础上初步探索了该树状化合物作为人呼吸道合胞病毒(Human respiratory syncytial virus, RSV)基因疫苗载体的在动物模型上应用。选取多个不同的电荷(N/P)比,制备PAMAM/DNA复合物,琼脂糖凝胶电泳结果表明,PAMAM G4及PAMAM G5都能够与DNA形成复合物。酶切保护实验结果表明,形成复合物后,DNA不被核酸酶降解,即PAMAM能够有效保护所携带的DNA分子不被降解。为进一步探索PAMAM的细胞毒性,分别选取了HEK293细胞、HepG2细胞、U251细胞及A549细胞进行毒性实验。结果表明,对于同种细胞系PAMAM G5的细胞毒性高于PAMAM G4的细胞毒性。PAMAMG4 48 h的半抑制率在0.1μg/μL,而PAMAM G5 48 h的半抑制率在0.05μg/μL。对于同一种PAMAM载体,随着浓度的增加毒性增大,细胞毒性呈现剂量依赖性。细胞凋亡实验结果表明,PAMAM在加入24 h后能够导致一些细胞坏死或晚期凋亡,早期凋亡现象不明显,同时,细胞坏死或晚期凋亡程度也呈现剂量依赖性,与MTT检测结果一致。在以上实验的基础上,选取N/P比为10：1作为免疫小鼠实验制备PAMAM/DNA复合物的标准。小鼠免疫实验初步结果表明经腿部肌肉注射PAMAM/DNA复合物,小鼠产生相应抗体。
[Abstract]:DNA vaccine can stimulate the production of cellular and humoral immunity, which is low cost, easy to store and transport. However, the naked DNA vaccine is easy to be hydrolyzed by enzyme during its transport in vivo. Can't cause enough immunogenicity. It is difficult to achieve good immuno-protective effect. DNA delivery vector can protect DNA molecule from hydrolysis by enzyme. In this study, polyamide-amine PamAm dendrimer was selected as gene vaccine delivery vector. The cytotoxicity of PAMAM G4 and PAMAM G5 in various cell lines was studied by cytotoxicity test and apoptosis test. On this basis, the dendritic compound was preliminarily explored as a human respiratory syncytial virus of human respiratory syncytial virus. The application of RSV-based gene vaccine vector in animal models. PAMAM/DNA complexes were prepared with different charge N / P ratios. The results of agarose gel electrophoresis showed that N- / P-. Both PAMAM G4 and PAMAM G5 could form complexes with DNA. In order to further explore the cytotoxicity of PAMAM, HEK293 cell line HepG2 cells were selected. The toxicity of U251 cells and A549 cells was tested. The cytotoxicity of the same cell line PAMAM G5 was higher than that of PAMAMG4. The half inhibition rate of PAMAMG4 for 48 h was 0.1 渭 g / 渭 L. The semi-inhibition rate of PAMAM G548 h was 0.05 渭 g / 渭 L. for the same PAMAM carrier, the toxicity increased with the increase of concentration. The results of apoptosis assay showed that PAMAM could lead to necrosis or late apoptosis of some cells after 24 h, but the early apoptosis was not obvious at the same time. The degree of cell necrosis or late apoptosis was also in a dose-dependent manner, which was consistent with the results of MTT. The N- / P ratio of 10: 1 was selected as the standard for the preparation of PAMAM/DNA complexes in mice. The preliminary results of mouse immunological tests showed that the PAMAM/DNA complexes were injected intramuscularly through the legs. The corresponding antibody was produced in mice.
【学位授予单位】：北京交通大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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