新型脂质-HAP-DNA复合体用于基因转染研究

发布时间：2018-08-05 19:23

【摘要】：基因治疗是将基因导入人体细胞中,以期产生有益的蛋白质来治疗疾病的一种方法。1995年,美国国立卫生组织(NIH)提出基因治疗目前存在的三大关键问题之重点问题便是:具有良好选择性、安全性和高效性的基因导入系统(即基因载体)。非病毒载体由于具有低毒,低免疫反应,靶向性和易于组装等优点,但转染效率一直不高。如何提高DNA在基因转染和基因治疗中的转染效率成为-基因转导技术的关键问题。因此寻找新的非病毒载体成为目前的研究热点。本研究自行组装构建了一种新型非病毒基因载体脂质-HAP-DNA复合体(Lipid—Hydroxyapatite—DNA,LHD)并验证其用作基因转导的可行性。本论文分为三个部分: 第一研究了HAP纳米粒子的制备与改性。通过该部分研究我们得到如下结论: 1.运用化学沉淀法合成了HAP纳米颗粒并进行了表面改性。得到了Ca/P比接近1.67的HAP纳米颗粒,在缓冲液中稳定性及分散性良好、粒径均匀、符合下一步研究需要。 2.利用有机大分子A作为稳定剂,改性后HAP表面Zeta电位为正,在缓冲液中可长时间稳定存在。 3.化学沉淀法合成纳米HAP时,反应温度、PH值对产物HAP的影响显著,溶液浓度对产物HAP影响不大。 4.超声处理、滴加速度及搅拌强度作为辅助手段会对制备所得纳米HAP晶体尺寸产生影响,而稳定剂种类和剂量会显著影响生成纳米HAP的表面特性。第二进行了LHD复合体的制备研究。得到如下结论: 1.用正电荷的纳米HAP分别与不同比例的DNA结合,最终确定b DNA与HAP的较为合适的结合比例。 2.用反相蒸发法将DNA—HAP复合物包裹进入脂质膜中,制备得到LHD复合体。观察到合成的LHD复合体成球形、具有一定包封率,并且对被包裹的DNA起到有效的保护作用。 3.在LHD复合体的制备中,进行了温度变化和光线照射对卵磷脂氧化率和LHD的包封率的影响的研究。确定了实验的防氧化措施及贮存方法。
[Abstract]:Gene therapy is a method of introducing genes into human cells in order to produce beneficial proteins to treat diseases. The National Health Organization (NIH) has put forward three key problems in gene therapy: a gene transfer system (gene vector) with good selectivity, safety and efficiency. Non-viral vectors have the advantages of low toxicity, low immune response, targeting and easy assembly, but the transfection efficiency is not high. How to improve the transfection efficiency of DNA in gene transfection and gene therapy has become a key issue in gene transduction technology. Therefore, the search for new non-viral vectors has become a hot research topic. A novel non-viral gene vector lipid-HAP-DNA complex (Lipid-Hydroxyapatite-DNA LHD) was constructed and its feasibility for gene transduction was verified. This thesis is divided into three parts: firstly, the preparation and modification of HAP nanoparticles are studied. Through this part of the study, we get the following conclusions: 1. HAP nanoparticles were synthesized and modified by chemical precipitation method. The HAP nanoparticles with Ca/P ratio close to 1.67 have good stability and dispersion in buffer solution, and the particle size is uniform, which meets the need of further study. Using organic macromolecule A as stabilizer, the surface Zeta potential of modified HAP is positive, which can be stabilized in buffer solution for a long time. In the synthesis of nanometer HAP by chemical precipitation method, the pH value of the reaction temperature has a significant effect on the product HAP, while the concentration of solution has little effect on the product HAP. Ultrasonic treatment, drop acceleration and stirring intensity as auxiliary means will affect the size of the prepared nanocrystalline HAP crystal, and the type and dosage of stabilizer will significantly affect the surface characteristics of the nanocrystalline HAP. Secondly, the preparation of LHD complex was studied. The conclusions are as follows: 1. The proper binding ratio of b DNA and HAP was determined by combining positive charge nanometer HAP with different proportion of DNA respectively. The DNA-HAP complex was encapsulated into the lipid membrane by reversed-phase evaporation to prepare the LHD complex. It is observed that the synthesized LHD complex is spherical, has a certain encapsulation efficiency and has an effective protective effect on the encapsulated DNA. In the preparation of LHD complex, the effects of temperature and light irradiation on the oxidation rate of lecithin and the encapsulation efficiency of LHD were studied. The anti-oxidation measures and storage methods are determined.
【学位授予单位】：武汉理工大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

【参考文献】