新型基因枪用羟基磷灰石微粒的研制

发布时间：2017-12-31 06:42

本文关键词：新型基因枪用羟基磷灰石微粒的研制　出处：《武汉理工大学》2010年硕士论文　论文类型：学位论文

【摘要】： 人类很多疾病是由基因上的缺陷所造成的,如果能从基因的水平上修复缺陷基因,将对传统疾病治疗方式产生革命性的影响。当前基因治疗中的基因递送系统主要分为病毒性载体和非病毒性载体两大类。病毒性载体在临床应用中具有潜在的危险性,而非病毒性载体转染效率较低。基因枪技术是一种纯物理的基因转移技术,相比之下有着明显的优势：操作简单、传递基因迅速、安全等,这一系列优点使其展现出诱人的应用前景,但基因枪技术中负载基因的金或钨不能被生命机体代谢,具有潜在的安全性。考虑到羟基磷灰石生物相容性好、可代谢、无细胞毒性等优点,本课题拟选用其作为金或钨的替代材料,并探讨其用于基因枪系统的有效性。实验研究主要包括以下三个部分：第一部分：稳定剂H保护法制备HAP微粒目的：制备出粒径均匀,分散性良好,具有特定表面性质及特定粒径范围的HAP微粒。方法：采用稳定剂H保护沉淀法制备HAP微粒,并进行粒度测试、电位分析和形貌测定,以研究不同反应条件对HAP表面特性的影响。结果：经Ca／P比值测定、电镜测试和粒度、电位测试,所得HAP微粒稳定,分散性良好,粒径小且均匀。结论：1、稳定剂H保护法制备的羟基磷灰石呈针状颗粒,粒径小且均匀。 2、稳定剂H用量、反应温度、Ca(OH)2滴加速度、超声处理等对产物粒径、电位均有一定影响。第二部分：pEGFP-C3质粒DNA的制备及HAP对DNA的吸附研究目的：制备出纯度、浓度都符合基因转染要求的pEGFP-C3质粒DNA,并将其较好的负载在HAP微粒上。方法：通过碱裂解法制备pEGFP-C3质粒DNA,并采用琼脂糖凝胶电泳和紫外吸收法对其进行浓度、纯度测定；然后采用紫外分光光度法测定不同操作条件对HAP吸附DNA的影响。结果：1、检测结果显示,制备得到的质粒纯度高,符合基因转染的要求。 2、不同的加样顺序及亚精胺的pH调节对DNA在HAP上的吸附容量影响甚微。结论：1、HAP吸附DNA时,可考虑加入亚精胺以增大其吸附容量。2、只要保证一定的HAP、DNA和亚精胺反应体系,均可获得较好的负载。第三部分：HAP纳米载体介导的RSC96细胞体外基因转染实验目的：摸索、研究羟基磷灰石纳米粒作为基因转移载体的有效性。方法：将制备得到的pEGFP-C3质粒作为目的基因,采用不同的微弹材料和不同的基因枪仪器参数进行转染,分析、比较其转染效果的差别。结果：1、金为载体时,视野中出现大量绿色荧光。2、HAP为载体时,视野中荧光量稍少。结论：金负载DNA制成基因微弹,用于基因枪系统,其转染效果明显。在一定的基因枪系统仪器参数条件下,HAP是能作为载体介导质粒DNA进入靶细胞内,并表达产生绿色荧光蛋白,以上说明HAP用于基因枪系统是可行的,但转染效率和金相比略低。
[Abstract]:Many human diseases are caused by genetic defects, if can repair the defective gene from the gene level, will have a revolutionary impact on the traditional disease treatment. The gene therapy of gene delivery system is mainly divided into viral vectors and non viral vectors. Two kinds of viral vectors is dangerous the potential in clinical application, and the transfection efficiency of non viral vector is low. Gene gun technology is a kind of pure physical gene transfer technology, by contrast has obvious advantages: simple operation, safety, rapid gene transfer, a series of advantages, showing an attractive prospect, but the gene gun technology supported gold or tungsten can not gene by life metabolism, with potential safety. Considering the hydroxyapatite has good biocompatibility, can metabolism, cell toxicity and other advantages, this paper intends to use it as An alternative material for gold or tungsten and the effectiveness of its use in the gene gun system. The experimental study mainly includes the following three parts:
The first part: the preparation of HAP particles by the stabilizer H protection method
Objective: to prepare HAP particles with uniform particle size, good dispersibility and specific surface properties and specific particle size range.
Methods: HAP particles were prepared by stabilizer H protection precipitation method, and particle size test, potential analysis and morphology measurement were carried out to study the effect of different reaction conditions on the surface characteristics of HAP.
Results: through the determination of Ca / P ratio, electron microscope test, particle size and potential test, the obtained HAP particles are stable, good dispersion, small particle size and uniform.
Conclusion: 1, the hydroxyapatite prepared by the stabilizer H protection method is acicular particles with small and uniform particle size.
2, the amount of stabilizer H, the reaction temperature, the 2 drop acceleration of Ca (OH), and the ultrasonic treatment have a certain effect on the particle size and potential of the products.
The second part: the preparation of pEGFP-C3 plasmid DNA and the study of the adsorption of DNA by HAP
Objective: to prepare the pEGFP-C3 plasmid DNA with the purity and concentration which are in line with the gene transfection requirement and load it on HAP particles.
Methods: pEGFP-C3 plasmid DNA was prepared by alkaline lysis, and its concentration and purity were determined by agarose gel electrophoresis and ultraviolet absorption. Then, the influence of different operation conditions on HAP adsorption on DNA was determined by ultraviolet spectrophotometry.
Results: 1, the results showed that the purity of the prepared plasmid was high, which was in line with the requirements of gene transfection.
2, the different addition order and pH regulation of spermidine have little influence on the adsorption capacity of DNA on HAP.
Conclusion: 1. When HAP adsorbed DNA, it could consider adding spermidine to increase its adsorption capacity.2. As long as a certain HAP was guaranteed, DNA and spermidine reaction system could get better load.
The third part: HAP nanoscale mediated gene transfection of RSC96 cells in vitro
Objective: To explore the effectiveness of hydroxyapatite nanoparticles as a gene transfer carrier.
Methods: the pEGFP-C3 plasmid was used as target gene, and different microprojectile materials and different gene gun parameters were used to transfect, and the difference of transfection efficiency was analyzed.
Results: 1, when gold is the carrier, a large number of green fluorescent.2 appears in the field of vision. When HAP is the carrier, the fluorescence in the field of vision is slightly less.
Conclusion: DNA gene into gold supported micro bomb for gene gun system, its effect is obvious. The transfection of gene gun system of certain parameters, HAP can be used as a carrier mediated plasmid DNA into target cells, and the expression of green fluorescent protein, HAP described above for gene gun system is feasible, but the transfection efficiency and metallographic slightly lower.

【学位授予单位】：武汉理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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