【作者】 蒙世芳；

【导师】 陈建海；

【作者基本信息】 南方医科大学 ， 药理学， 2010， 硕士

【摘要】 背景：随着近代生物技术的发展与人类基因库的不断完善,人们在理论上和实践上的不断探索,基因治疗对许多疾病提供了一个新的治疗途径。所谓基因治疗是指在基因水平上将有功能的基因或其他基因通过基因转移方式导入患者体内,并使之表达正常的基因,或表达患者原来不存在或表达很低的外源基因。基因治疗的基本条件包括：目的基因的获取,靶细胞的选择,将目的基因导入靶细胞的方法和途径。目前基因载体的主要分为两类：病毒载体或非病毒载体。非病毒载体具有的安全性,低免疫原性等优势成为研究的重点。其中如何将目的基因有效的转运到靶细胞或者靶部位是十分关键的步骤,基因枪作为纯物理方式的非病毒转染方法能够方便、有效的转运基因,已经越来越受到研究者的关注。目的：合成葡聚糖-精胺阳离子聚合物DSP,扩增和提取质粒DNApmcherry-c1和pEGFP-c3,研究以DSP为粘合剂制备基因枪子弹的方法,初步探讨在同等条件下,用基因枪方法和普通转染方法在体外的转染能力比较,和用DSP作为粘合剂和用传统亚精胺作为粘合剂进行体外转染能力的比较,以及用基因枪方法共转染同单独转染能否发挥协同作用,提高转染率的研究。方法：研究首先以平均分子量为40KDa的葡聚糖作为反应起始物,在室温下经高碘酸钾氧化6个小时,得到了氧化葡聚糖。通过盐酸羟胺法测定了氧化葡聚糖醛基的含量,根据此含量按照1：1.25摩尔比的精胺与氧化葡聚糖反应,该反应能生成含希夫氏碱的葡聚糖-精胺亚胺聚合物,所得到的聚合物在过量硼氢化钠作用下,可还原生成稳定葡聚糖—精胺共价阳离子聚合物(DSP)。DSP通过FT-IR及1H-NMR其结构进行鉴定,并用三硝基笨磺酸法(TNBS)测定其醛基的含量。采用碱裂解法分别扩增和提取了具有红色荧光蛋白的质粒pmcherry-c1和具有绿色荧光蛋白的质粒pEGFP-c3。首先将质粒DNA转入到大肠杆菌中,增殖转化的大肠杆菌扩增质粒DNA,然后采用碱裂解法从大肠杆菌中提取质粒DNA并采用质粒提取试剂盒除去其中的菌体残片及内毒素等进行了纯化。通过紫外分光光度计测量其浓度和纯度,并用琼脂糖凝胶电泳进行了鉴定。制备了基因枪子弹,以DSP代替传统的亚精胺做为粘合剂制备基因枪子弹,以pmcherry-c1为报告基因,轰击乳腺癌细胞(MCF-7),考察了基因枪转染技术对细胞的转染率和细胞的生存率的影响,并采用了星点设计-效应面法优化了试验的条件,通过荧光倒置显微镜观察细胞的转染情况并计算转染率；MTT法考察复合物细胞毒性,在同等条件下以亚精胺法制备了基因枪子弹作为对照。在此基础上,研究了以DSP为粘合剂制备的基因枪子弹实现了两种质粒pmcherry-c1和PEGFP-c3的共转染,以亚精胺法制备了基因枪共转染子弹作为对照。结果：所合成氧化葡聚糖醛基含量为7.27 mmol/g, DSP中接枝精胺的浓度为0.0214mg/ml。所提取的质粒DNA的纯度(比值在1.8-2.0之间)pmcherry-cl A260/A280=1.94, pEGFP-c3 A260/A280=1.92,其浓度分别Cpmcherry-c1= 492.8ug/ml, CpEGFP-c3=498.1ug/ml.用DSP为载体,用化学方法转染质粒pmcherry-c1,得到转染率为14.8%,细胞生存率为83.2%。用所合成的DSP代替传统的亚精胺作为粘合剂,以质粒pmcherry-c1作为报告基因,制备了基因枪子弹,用星点设计-效应面法,优化了用基因枪法转染体外MCF-7细胞转染条件。其优化条件为：每发子弹含钨粉的量为1.0mg,质粒DNA的量为1.2ug,基因枪的压力为1.5kpa,轰击距离2cm。得到的转染效率为22.1%,细胞生存率为52.2%,同通过模型预测值的偏差的绝对值为8.3%,6.1%。同时在同等条件下,用传统的亚精胺法制备了基因枪子弹作为对照,轰击细胞后得到其转染率为15.8%,细胞生存率为54.5%。研究了以DSP为粘合剂制备的基因枪子弹实现了2种质粒pmcherry-c1和pEGFP-c3的共转染,在每发子弹含钨粉的量为1.0mg,质粒DNA的量为1.2ug,基因枪的压力为1.5kpa,轰击距离2cm条件下,单独转染质粒pmcherry-c1得到转染率为21.0%,单独转染质粒pEGFP-c3得到转染率为19.9%,共同转染这两种质粒DNA得到转染率为23.0%,同时,以同样方法下以亚精胺法制备了基因枪,在同等条件下转染细胞作为对照,单独转染质粒pmcherry-c1得到转染率为15.5%,单独转染质粒pEGFP-c3得到转染率为15.2%,共同转染这两种质粒DNA得到转染率为17.9%。结论：该研究表明：用DSP制备的基因枪子弹比用DSP作为基因载体的化学方法体外细胞转染有一定的提高,比用传统的亚精胺法体外细胞转染也有一定的提高,并且用DSP制备的共转染比用传统的亚精胺法制备的共转染体外细胞转染也有一定的提高。采用星点设计-效应面法优化实验条件,能够很好的描述因素和效应之间的关系,方法确实可行。所合成的葡聚糖-精胺阳离子聚合物是一种制备工艺简单、低毒的高分子化合物,并可以代替亚精胺制备基因枪子弹。更多还原

【Abstract】 Background:With the development of modern biotechnology and the improvement of the human gene pool, gene therapy offers a new approach for treatment of many human diseases after the exploration in the theory and practice. Gene therapy is an approach for treatment of diseases by inserting functional gene or other gene into patients in order to produce normal, none or low expression of exogenous gene. The basic criteria for gene therapy is the acquisition of the targeted gene, the selection of targeted cells and the delivery system of the targeted gene into the targeted cells. Gene delivery systems are classified into viral and non-viral systems. The non-viral system is an very important system for its safety and low immunogenicity. But how to improve the transfection efficiency is a very difficult problem. In this study we choose the gene gun as an delivery system in order to offer a convenient and efficient way.Objective:To synthesize dextran-spermine polycation (DSP)as a gene vector, amplifly and extract plasmid DNA pmcherry-cl and pEGFP-c3, investigate the methods of preparation of gene gun bullets based on DSP as gluing agent, compare with the gene gun and ordinary delivery systems of the transfection efficiency in vitro, compare with the DSP and spermidine as gluin agent effecting on the transfection efficiency in vitro, and evaluate the gene gun whether or not enhance the transfection efficiency.Methods:40 KDa Dextran was oxidized under room temperature for 6h using potassium periodate to obtain oxidized dextran, which was then reacted with spermine(1 to 1.25 mole ratio)to form imine-based polycation. The Schiff base polycation was reduced to the stable amine-based dextran-spermine polycation by sodium borohydride. The total nitrogen and primary amine content were determined by elemental analysis and TNBS, respectively. The chemical structure of DSP was elucidated by 1H-NMR and FT-IR.Plasmid pmcherry-cl with red fluorescent protein and plasmid pEGFP-c3 with green fluorescent protein was ampliflied and extracted by alkaline lysis. Firstly, the plasmid were inserted into the E.coli for amplifling, and then were extracted by alkaline lysis from the E.coli and purified using plasmid extraction kit in order to get rid of the cell debris and endotoxin, eventually measured by UV and identified by agarose gel electrophoresis (AGE).The gene gun bullets which used pmcherry-cl as reported gene were based on DSP as gluing agent instead of spermidine and breast cancer cells (MCF-7) as the targeted cells, and the transfection efficiency were examined by fluorescence microscopy after transfection, investigated the influence on the cell transfection efficiency and cell survival rate of the gene gun-mediated gene transfection, simultaneously the central composite design-response surface methodology was used to optimize the conditions of delivery. The cytotoxicity of polyplexes was evaluated by MTT assay, with the spermidine for the control. Further more, the gene gun bullets constituted of DSP were cotransfected with plasmid pmcherry-cl and PEGFP-c3 compared with that of constituted of spermidine.Results:The aldehyde content of oxidized dextran was 7.27 mmol/g and the content of grafting of DSP was 0.0214mg/ml.The purity (ratio between 1.8 to 2.0) and the content of plasmid DNA pmcherry-cl and pEGFP-c3 (A260/A280) was 1.94,1.92, 492.8ug/ml,498.1ug/ml,respectively.The transfection efficiency and cell survival rate was 14.8% and 83.2% which based on DSP as gluing agent and transferred by chemical methods. The preparation of gene gun bullets used the plasmid pmcherry-cl as reported gene and the synthetized DSP as gluing agent instead of spermidine. The transferred conditions of gene gun- mediated gene transfection were optimized by the central composite design-response surface methodology, the results were:the content of tungsten in each bullet was 1.0mg; the content of plasmid DNA was 1.2ug; the pressure of the gene gun was 1.5kpa; the gene gun bombardment distance was 2cm. Based on these conditions, the transfection efficiency and the cell survival rate was 22.1% and 52.2% which with the absolute deviation of 8.3% and 6.1% compared with predicted model value. In the same conditions, the transfection efficiency and the cell survival rate based on spermidine as the gluing agent was 15.8% and 54.5%,respectively.the transfection efficiency of plasmid pmcherry-cl and pEGFP-c3 was 21.0% and 19.9%,and the cotransfection efficiency of the plasmid pmcherry-cl and pEGFP-c3 that prepared by DSP was 23.0%.However,the transfection efficiency of plasmid pmcherry-cl and pEGFP-c3 that prepared with spermidine was only 15.5% and 15.2%,and the cotransfection efficiency was 17.9%.Conclusion:This research indicates that the gene gun bullets prepared with DSP showed a better transfection efficiency in vitro compared with that prepared by spermidine. and chemical method Further more, the cotransfection with plasmid pmcherry-cl and pEGFP-c3 that prepared with DSP was also better than that prepared with spermidine. The central composite design-response surface methodology was well applied to the optimization of the conditions of delivery, it was feasible for description of the relationship between the factors and effects.The synthesized dextran-spermine polycation (DSP) was easily prepared as the gluing agent of gene gun bullets and shows low toxicity.更多还原