基于芳香共轭的两亲自组装肽衍生物的设计、合成及其转染能力研究

发布时间：2018-06-18 01:58

本文选题：非病毒载体 + π-π堆积　；参考：《吉林大学》2014年硕士论文

【摘要】：基因治疗作为一种新型的治疗手段，对常规治疗手段无法有效应对的疑难疾病（如肿瘤、免疫缺陷病及某些遗传病）有良好的临床应用前景。为实现基因治疗的目的，承载治疗基因的核酸（DNA或RNA）必须能够准确、稳定、高效地进入目的细胞，并且在病理细胞及组织中正常表达，发挥治疗作用。人体内环境的生理生化条件较为复杂，治疗用核酸如果暴露在其中，很可能出现不同程度的降解，进而无法达成预期的治疗效果。因此，寻求便利有效的基因传递手段成为了基因治疗应用于临床的首要任务之一。基因传递媒介主要分为物理手段、病毒载体和非病毒载体三大类。其中非病毒载体由于容量限制小、结构多样、安全可控、毒性低、生物相容性好、可降解等优点受到科研领域的关注。非病毒载体需要具备以下特点：容易包装治疗基因形成稳定的复合体，能有效进入靶细胞，在细胞内能迅速崩解释放核酸，无明显的生理或遗传毒性。目前商品化的转染试剂大多基于阳离子脂质体或阳离子聚合物，其进入细胞的途径是胞吞作用或阳离子逃逸效应，对细胞膜表现出了一定程度的损伤。而且其设计缺乏针对特定靶细胞的指向性，，这意味着在临床应用中这类试剂的治疗效果很大程度上会依赖于剂量与给药频度，治疗过程中很可能产生严重的副作用。因此，本文设计了一种利用短肽衍生物，利用其自组装形成的纳米结构作为基因载体，该载体系统具有较高的核酸搭载效率，细胞毒性低于常规转染试剂，同时有促进基因靶向传递的可能性。本文短肽衍生物FGPep的序列设计源于π-π堆积自组装的典型基序之一Fmoc-Phe-Phe，C端选取了针对HER2进行肽库筛选获得的配体序列之一(YSSPTQR)，以若干个丙氨酸残基作为两者的连接部位，使其亲水端具有HER2靶向性。其序列为Fmoc-FFAAYSSPTQR-NH2。我们采用经典的Fmoc固相肽合成法合成了该衍生肽，以HPLC对其进行纯化，目的产物收率达80%以上。FGPep能够在超声作用下在水相中均匀分散并自组装，形成有序的纳米线。通过琼脂糖凝胶电泳评估了FGPep包覆核酸的能力，在一定的浓度与混合比下，FGPep凝胶能够对所用质粒达到完全截留。选用SK-OV-3细胞作为考察衍生肽复合物细胞毒性的对象。当FGPep在培养基中的分散浓度达到500μM时，仍未见细胞有明显的凋亡，我们认为FGPep呈现的低细胞毒性符合其作为转染载体的要求。然而实际的转染效果不够理想。在同等pEGFP-N1加入量的条件下，实验组的目的蛋白GFP表达量远低于使用脂质体2000的阳性对照组。我们推测可能是FGPep与核酸形成的复合物尺寸偏大，难以诱导胞吞作用，导致细胞的核酸摄入量低下。综上所述，本文基于新思路设计的衍生肽具有自组装和包覆核酸的能力，而且其细胞毒性较低，安全性好。但距离作为转染材料的实际应用仍有一定差距，需要进一步研究与改进。
[Abstract]:As a new therapeutic method, gene therapy has a good prospect in clinical application for difficult diseases (such as tumor, immunodeficiency disease and some hereditary diseases) which can not be effectively dealt with by conventional therapy. In order to achieve the purpose of gene therapy, nucleic acid DNA or RNAs carrying therapeutic genes must be able to enter the target cells accurately, stably and efficiently, and express them normally in pathological cells and tissues to play a therapeutic role. The physiological and biochemical conditions of human body environment are more complex. If therapeutic nucleic acid is exposed to it, it may be degraded to different degrees, and the expected therapeutic effect can not be achieved. Therefore, it is one of the most important tasks for gene therapy to seek convenient and effective means of gene transmission. Gene transfer vectors are divided into three categories: physical means, viral vectors and non-viral vectors. Among them, non-viral vectors have attracted much attention due to their small capacity limitation, diverse structure, safe and controllable, low toxicity, good biocompatibility, biodegradability and so on. Non-viral vectors need to possess the following characteristics: it is easy to package therapeutic genes to form stable complexes, which can effectively enter target cells, rapidly disintegrate and release nucleic acids in cells without obvious physiological or genetic toxicity. At present, most of the commercial transfection reagents are based on cationic liposomes or cationic polymers, which enter cells through cytosolic action or cationic escape effect, showing a certain degree of damage to cell membrane. Moreover, its design is not directed to specific target cells, which means that the therapeutic effect of these reagents in clinical application depends largely on the dosage and frequency of administration, and the treatment process is likely to have serious side effects. In this paper, we designed a novel nucleic acid carrier using short peptide derivatives and its self-assembled nanostructures as gene carriers. The carrier system has higher nucleic acid carrying efficiency and lower cytotoxicity than conventional transfection reagents. At the same time, it is possible to promote gene targeting transmission. In this paper, the sequence design of short peptide derivative FGPep originated from Fmoc-Phe-Phe-Phe-Phe-Phe-C terminal, which is one of the typical motifs of 蟺-蟺 stacking self-assembly. One of the ligand sequences obtained by peptide library screening for HER2, YSSPTQRN, was selected, and several alanine residues were used as the connecting sites. So that the hydrophilic end of HER2 target. Its sequence is Fmoc-FFAAYSSPTQR-NH2. The derivative peptide was synthesized by the classical Fmoc solid phase peptide synthesis method and purified by HPLC. The yield of the derivative peptide was over 80%. FGPep could be uniformly dispersed and self-assembled in aqueous phase under ultrasonic irradiation to form ordered nanowires. The ability of FGPep to coat nucleic acid was evaluated by agarose gel electrophoresis. SK-OV-3 cells were selected to study the cytotoxicity of derived peptide complexes. When the concentration of FGPep in the medium reached 500 渭 M, there was no obvious apoptosis. We think that the low cytotoxicity of FGPep meets the requirement of FGPep as a transfection vector. However, the actual transfection effect is not ideal. Under the same dosage of pEGFP-N1, the expression of target protein GFP in the experimental group was much lower than that in the positive control group using liposome 2000. We speculate that the complex formed by FGPep and nucleic acid is too large to induce cytosptosis, resulting in a low intake of nucleic acid. To sum up, the derived peptides designed based on the new idea have the ability of self-assembly and nucleic acid coating, and their cytotoxicity is low and their safety is good. However, there is still a gap in the application of distance as a transfection material, which needs further study and improvement.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R914

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