聚乙烯亚胺纳米基因载体的实验研究
发布时间：2019-06-08 09:22
【摘要】： 基因治疗有三个重要环节,即目的基因、转基因载体和靶细胞。基因导入系统是基因治疗的核心技术。目前,应用于基因治疗的载体主要有病毒载体系统和非病毒载体系统。病毒载体转染效率高,是体内基因治疗的主要工具,但安全性存在隐患及有免疫原性,体内不能反复应用。而非病毒载体具有安全性高、免疫原性低、易于对DNA进行操作等优点,故人们愈来愈重视人工合成的非病毒载体的研究,而解决转染效率问题是非病毒中载体中最为关注的问题,理想的载体系统是能将治疗基因输送到并进入特定的靶细胞,从而能在该细胞中得到有效表达,这对于恶性肿瘤基因治疗尤为重要。以受体靶向的非病毒载体系统是最常用和有效的策略。利用细胞表面表达特异性的受体或蛋白,将特定的配体分子或片段与载体连接形成分子偶联体,使DNA能靶向性地转到表达受体的细胞。同时,针对非病毒载体的缺陷及DNA转移过程中的内吞小泡释放问题、转运入核问题,根据具体情况选择合适的载体,并对其作进一步的优化、改善以获得满意的治疗或应用效果。目前常用的非病毒载体包括裸DNA脂质体载体及阳离子多聚物型载体等。 纳米粒材料的选择是成功进行纳米基因转运和治疗的关键。所选择的材料必须是生物可降解型或者易于从体内排泄,而不产生有害的降解产物,且无免疫原性,不引起机体的免疫排斥反应。高分子生物降解材料制备的纳米颗粒具有稳定、无毒、无抗原性、生物相容性好、对所转运基因的表达有缓释作用及对基因有保护作用等优点,是良好的纳米基因转运载体材料。聚乙烯亚胺(polyethylenimine,PEI)是最常用的阳离子多聚物非病毒载体,PEI可把质粒DNA缩合(condense)成数百纳米大小的颗粒,通过静电作用吸附到细胞表面上,被动内吞。PEI在吞噬泡内不能降解,同时保护DNA免受溶酶体降解;另外,PEI有渗透性肿胀效应,导致吞噬泡破裂,使DNA进入胞浆,并促进DNA进入细胞核。 在本研究中,我们选择聚乙烯亚胺(polyethylenimine,PEI)通过自由基聚合法来制备PEI纳米粒(nanoparticles)作为基因转运载体。PEI纳米粒由于表面带正电荷而获得结合带负电DNA的能力及某些胶体特性。同时,实验也证实,PEI纳米颗粒/DNA复合物能保护所携带的DNA免受核酸酶的降解。当PEI纳米颗粒浓度在＜10mg/ml时PEI纳米颗粒对正常的肝细胞(L-02)以及HepG2细胞无细胞毒性,当PEI纳米颗粒浓度＞25 mg/ml时细胞毒性显著增加。在体外基因转运中,该纳米颗粒可有效转运EGFP(绿色荧光蛋白)报导基因表达质粒进入NIH3T3细胞,通过流式细胞仪的检测,PEI纳米粒转运效率达33%,强于相同条件下SuperFect~(TM)的转染效率。综上,PEI纳米载体是一种具备良好的生物安全性,高转染及靶向能力的基因载体系统。
[Abstract]:Gene therapy has three important links, namely, target gene, transgenic vector and target cell. Gene introduction system is the core technology of gene therapy. At present, the main vectors used in gene therapy are viral vector system and non-viral vector system. Virus vector transfer efficiency is high, is the main tool of gene therapy in vivo, but the safety has hidden dangers and immunogenicity, and can not be used repeatedly in vivo. However, non-viral vectors have the advantages of high safety, low immunogenicity and easy to operate DNA, so people pay more and more attention to the research of synthetic non-viral vectors. To solve the problem of transfer efficiency is the most concerned problem in the non-viral vector. The ideal vector system is that the therapeutic gene can be transported to and into a specific target cell, so that it can be effectively expressed in the cell. This is particularly important for gene therapy of malignant tumors. Receptor targeting non-viral vector system is the most commonly used and effective strategy. A specific receptor or protein was expressed on the cell surface, and a specific ligand molecule or fragment was connected with the vector to form a molecular coupling, so that DNA could be targeted to the cell expressing the receptor. At the same time, aiming at the defects of non-viral vectors and the release of endocytosis vesicles in the process of DNA transfer, the appropriate vectors were selected according to the specific conditions, and the appropriate vectors were further optimized. Improve to achieve satisfactory treatment or application results. At present, the commonly used non-viral vectors include naked DNA liposomes vector and cationic polymer vector. The selection of nanoparticles is the key to successful gene transfer and treatment. The selected material must be biodegradable or easily excreted from the body without harmful degradation products, no immunogenicity, and no immune rejection. The nanoparticles prepared by polymer biodegradable materials have the advantages of stability, non-toxicity, no antigenicity, good biocompatibility, sustained release effect on the expression of transporter genes and protective effect on genes. It is a good nano-gene transport vector material. Polyethylene imine (polyethylenimine,PEI) is the most commonly used cationic polymer non-viral vector. PEI can condensation the plasmid DNA into hundreds of nanometers of particles, which can be adsorbed on the cell surface by electrostatic action. Passive endocytosis. PEI could not be degraded in phagocytic vesicles and protected DNA from lysosome degradation. In addition, PEI has osmotic swelling effect, which leads to the rupture of phagocytic vesicles, the entry of DNA into the cytoplasm, and the promotion of DNA into the nucleus. In this study, we selected polyethylene imine (polyethylenimine,). PEI nanoparticles (nanoparticles) were prepared by free radical polymerization as gene transfer vector. PEI nanoparticles obtained the ability of binding negative DNA and some colloidal properties because of the positive charge on the surface. At the same time, it was also confirmed that PEI nanoparticles / DNA complex could protect the DNA from the degradation of nuclease. When the concentration of PEI nanoparticles was < 10mg/ml, PEI nanoparticles had no cytotoxicity to normal hepatocytes (L 鈮，
本文编号：2495181
[Abstract]:Gene therapy has three important links, namely, target gene, transgenic vector and target cell. Gene introduction system is the core technology of gene therapy. At present, the main vectors used in gene therapy are viral vector system and non-viral vector system. Virus vector transfer efficiency is high, is the main tool of gene therapy in vivo, but the safety has hidden dangers and immunogenicity, and can not be used repeatedly in vivo. However, non-viral vectors have the advantages of high safety, low immunogenicity and easy to operate DNA, so people pay more and more attention to the research of synthetic non-viral vectors. To solve the problem of transfer efficiency is the most concerned problem in the non-viral vector. The ideal vector system is that the therapeutic gene can be transported to and into a specific target cell, so that it can be effectively expressed in the cell. This is particularly important for gene therapy of malignant tumors. Receptor targeting non-viral vector system is the most commonly used and effective strategy. A specific receptor or protein was expressed on the cell surface, and a specific ligand molecule or fragment was connected with the vector to form a molecular coupling, so that DNA could be targeted to the cell expressing the receptor. At the same time, aiming at the defects of non-viral vectors and the release of endocytosis vesicles in the process of DNA transfer, the appropriate vectors were selected according to the specific conditions, and the appropriate vectors were further optimized. Improve to achieve satisfactory treatment or application results. At present, the commonly used non-viral vectors include naked DNA liposomes vector and cationic polymer vector. The selection of nanoparticles is the key to successful gene transfer and treatment. The selected material must be biodegradable or easily excreted from the body without harmful degradation products, no immunogenicity, and no immune rejection. The nanoparticles prepared by polymer biodegradable materials have the advantages of stability, non-toxicity, no antigenicity, good biocompatibility, sustained release effect on the expression of transporter genes and protective effect on genes. It is a good nano-gene transport vector material. Polyethylene imine (polyethylenimine,PEI) is the most commonly used cationic polymer non-viral vector. PEI can condensation the plasmid DNA into hundreds of nanometers of particles, which can be adsorbed on the cell surface by electrostatic action. Passive endocytosis. PEI could not be degraded in phagocytic vesicles and protected DNA from lysosome degradation. In addition, PEI has osmotic swelling effect, which leads to the rupture of phagocytic vesicles, the entry of DNA into the cytoplasm, and the promotion of DNA into the nucleus. In this study, we selected polyethylene imine (polyethylenimine,). PEI nanoparticles (nanoparticles) were prepared by free radical polymerization as gene transfer vector. PEI nanoparticles obtained the ability of binding negative DNA and some colloidal properties because of the positive charge on the surface. At the same time, it was also confirmed that PEI nanoparticles / DNA complex could protect the DNA from the degradation of nuclease. When the concentration of PEI nanoparticles was < 10mg/ml, PEI nanoparticles had no cytotoxicity to normal hepatocytes (L 鈮，
本文编号：2495181
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