硅纳米颗粒作为基因转染载体的研究

通过不同浓度的NaCl、NaI修饰硅纳米颗粒,用琼脂糖凝胶电泳分析硅纳米颗粒与DNA结合力及对DNA的保护作用,同时用绿色荧光蛋白基因作报告基因,以硅纳米颗粒作为基因转染的载体,转染HT1080细胞。经电镜观察证实硅纳米颗粒进入细胞内;硅纳米颗粒与DNA结合后,能对DNA起保护作用;并且硅颗粒作为基因转染的载体,将绿色荧光蛋白基因导入HT1080细胞,用荧光显微镜观察到发绿色荧光的细胞。结果表明,硅纳米颗粒可作为基因转染的载体。     

磁性纳米颗粒作为基因转染载体的研究

通过扫描电子显微镜和Zeta电位仪对磁性纳米颗粒的形貌、粒径、表面电位等进行了表征。利用凝胶电泳阻滞试验分析磁性纳米颗粒与DNA的结合情况,研究磁性纳米颗粒对DNA的保护效果,运用MTT和流式细胞术分析磁性纳米颗粒对细胞的毒性。以绿色荧光蛋白基因为报告基因进行293T细胞的转染,研究磁性纳米颗粒与质粒DNA不同比例条件下对293T细胞的转染效率,并与脂质体(Lipofectamine2000)介导的转染进行比较分析。结果表明,磁性纳米颗粒与DNA可以稳定结合,可以保护DNA免受酶的消化作用,当磁性纳米颗粒与DNA比为1 1时,转染效率最高,优于脂质体(Lipotamine2000)介导的转染,且对细胞的毒害作用小于Lipotamine2000。     

纳米颗粒作为基因载体的应用

随着纳米技术的发展,纳米颗粒因具有较高的转染效率、良好的靶向性及有效的基因保护作用而被用作基因载体。简要介绍了磁性纳米颗粒、硅纳米颗粒及阳离子多聚物颗粒等的研究进展。     

猪肾细胞脂质磁转染系统的构建与表征

磁性纳米基因载体是一种非病毒基因载体,经过功能性基团修饰后能够连接阳离子转染剂构建细胞转染系统。本文将磁转染技术结合常用的脂质体转染,形成了一种新型动物体细胞转染方法,即称脂质磁转染(Liposomal magnetofection,LMF)。这将为体细胞克隆培育转基因动物提供稳定遗传的细胞系。为构建脂质磁性纳米基因载体复合物系统,本研究利用一种磁性纳米基因载体通过分子自组装与脂质阳离子转染剂结合,用于携带外源基因转染动物体细胞。通过原子力显微镜(AFM)观测、ζ电位-粒度等分析表征手段,研究磁性纳米基因载体的形貌、粒径分布、负载及浓缩DNA的方式。结果表明,通过猪肾(PK)细胞的LMF实验,与脂质体(Lipofectamine2000)介导的转染比较,具有较高的转染率,更重要的是克服了脂质体转染瞬时表达的缺陷。MTT细胞毒性试验结果也显示该方法具有较低的细胞毒性。因此LMF是一种切实可行的高效低毒性的细胞转染方法。     

纳米基因载体在植物遗传转化中的应用

纳米基因载体已成功地应用于生物医学领域并显示了优越的转染效率、良好的生物相容性和有效的基因保护作用。近年来,纳米颗粒作为基因载体在植物转导中的应用潜力越来越受到关注,也为植物遗传工程提供了新的可能性。在阐述纳米载体的特性、在植物细胞中的转导机制及转导优势的基础上,重点讨论了纳米载体在植物转基因中的应用,并对其前景进行了展望。     

壳聚糖纳米粒介导基因转染的影响因素及改性修饰

壳聚糖带正电荷,可与带负电荷的DNA结合形成纳米级的多聚复合物(纳米粒)。作为一种基因载体,壳聚糖对DNA具有很好的结合和保护作用,对生物体无毒、相容性好,被广泛应用于基因转染及基因预防和治疗中。壳聚糖的主要缺点是转染效率较低,但对其进行改性或修饰后,有可能提高其转染效率。     

量子点在基因转染中的应用研究进展

量子点因其独特的纳米尺寸效应、光学特性和生物相容性,既能作为纳米载体与目的基因结合,又能作为纳米荧光标记物跟踪记录其在转染过程中的位置,给基因工程的发展带来了新的契机。在阐述量子点用于基因转染的优势、标记基因的方法等基础上,作者系统综述了量子点在基因转染中的应用,并对其发展趋势和应用前景进行了展望。     

基于纳米基因载体的动植物遗传转化研究进展

转基因技术在动植物优良新品种的培育中发挥着重要作用,而随着纳米生物技术的发展,基于纳米材料构建基因载体的动植物转基因技术,对于发展动植物转基因新方法以及加速转基因种质材料的大规模制备、优良新品种的培育进程具有更为重要的意义。综述了纳米基因载体的种类与性质,并结合动植物遗传育种的研究进展,分析了纳米基因载体相比于其他载体的特点及优势,同时,重点阐述了基于纳米基因载体的基因转染技术的基本原理和操作过程,及其在动植物遗传转化中的应用,以期为动植物基因工程改造提供新思路。     

聚乙烯亚胺转基因影响因素的测定及其优化

聚乙烯亚胺 (PEI)为阳离子多聚物 ,可浓缩DNA形成纳米级颗粒 ,作为基因释放载体转染真核细胞 .选用Mr2 5 0 0 0 ,分枝状的聚乙烯亚胺转染质粒 ,比较多种转基因效率的影响因素 .通过MTT法测定PEI对COS 7细胞的细胞毒性 .利用电泳阻滞实验测定PEI与DNA形成复合物时所需的比例 .通过PEI转染增强型绿色荧光蛋白的pEGFP质粒、编码β 半乳糖苷酶的pSVβ质粒 ,探索氯喹、白蛋白、血清、盐离子浓度、质粒剂量、细胞数量等对聚乙烯亚胺转基因效率的影响 .实验发现 ,PEI对细胞的毒性作用与剂量相关 .PEI DNA的N P比在 3 0以上方可完全结合DNA .溶酶体抑制剂氯喹可增加转染效率 .培养液中的白蛋白、血清会降低转染效率 .生理盐溶液作为配制PEI DNA复合物的溶媒 ,转染效率高于 5 %葡萄糖作为溶媒 .随着转染质粒剂量的增加 ,转染效率呈剂量依赖正效应 .聚乙烯亚胺是有效的体外真核细胞转染剂 ,可用于合成更复杂的基因释放载体 .     

两种阳离子纳米基因载体及植物基因介导效果的研究

以阳离子聚乙烯亚胺(polyethylenimine, PEI)和壳聚糖(chitosan, CS)作为两种植物基因载体,分别制备了载基因PEI纳米粒(PEI/DNA)和壳聚糖-DNA纳米粒(CS/DNA),并对其形态、粒度分布、包封率、DNA结合的稳定性及纳米颗粒对DNA的保护等方面进行表征.并以GFP基因为报告基因进行植物细胞转染,比较两者转化效率.结果表明PEI/DNA纳米粒稳定性,对DNA的保护以及转染效率等方面均优于壳聚糖-DNA纳米粒.     

Magnetic nanoparticles with surface modification enhanced gene delivery of HVJ-E vector

To enter the realm of human gene therapy, a novel drug delivery system is required for efficient delivery of small molecules with high safety for clinical usage. We have developed a unique vector "HVJ-E (hemagglutinating virus of Japan-envelope)" that can rapidly transfer plasmid DNA, oligonucleotide, and protein into cells by cell-fusion. In this study, we associated HVJ-E with magnetic nanoparticles, which can potentially enhance its transfection efficiency in the presence of a magnetic force. Magnetic nanoparticles, such as maghemite, with an average size of 29 nm, can be regulated by a magnetic force and basically consist of oxidized Fe which is commonly used as a supplement for the treatment of anemia. A mixture of magnetite particles with protamine sulfate, which gives a cationic surface charge on the maghemite particles, significantly enhanced the transfection efficiency in an in vitro cell culture system based on HVJ-E technology, resulting in a reduction in the required titer of HVJ. Addition of magnetic nanoparticles would enhance the association of HVJ-E with the cell membrane with a magnetic force. However, maghemite particles surface-coated with heparin, but not protamine sulfate, enhanced the transfection efficiency in the analysis of direct injection into the mouse liver in an in vivo model. The size and surface chemistry of magnetic particles could be tailored accordingly to meet specific demands of physical and biological characteristics. Overall, magnetic nanoparticles with different surface modifications can enhance HVJ-E-based gene transfer by modification of the size or charge, which could potentially help to overcome fundamental limitations to gene therapy in vivo.     

葡聚糖磁性纳米颗粒外加磁场对人树突状细胞基因转染效率的研究

目的研究葡聚糖磁性纳米颗粒（the dextran coated magnetic iron oxide nanoparticles,DMN）在外加钕一铁一硼稀土固定磁场的作用下对人树突状细胞转染效率以及安全性的影响。方法先通过磁力计对DMN进行分析;再将修饰有多聚赖氨酸（Poly-L—Lysine,PLL）的DMN携带绿色荧光蛋白pEGFP—Cl质粒报告基因,在钕-铁-硼稀土周定强磁场的作用下,体外转染人树突状细胞,用荧光显微镜直接观察和流式细胞仪检测来评价外加磁场对DMN作为人树突状细胞转染载体效率的影响;在转染后采用MTT比色法测定在磁场干预下的DMN对人树突状细胞增殖和功能的影响以了解其细胞毒性。结果DMN的核心直径〈30nm,具有明硅的超顺磁性,比饱和磁化强度也明显高于相同Fe3O4含量的普通磁块;DMN作为基因载体在外加磁场作用下,转染12h即可将报告基因转染至人树突状细胞内并成功表达,在荧光显微镜下可观察到绿色荧光细胞,24h转染率可达到最高（约为27％）,转染效率较未加磁场组提高了2～4倍。而且转染后的人树突状细胞增殖活性及功能未因DMN外加磁场及其作用时间的长短而受到影响。结论超顺磁性的DMN在外加磁场作用下可以明显、安全、有效地提高对人树突状细胞的转染效率。     

Generation of magnetic nonviral gene transfer agents and magnetofection in vitro

This protocol details how to design and conduct experiments to deliver nucleic acids to adherent and suspension cell cultures in vitro by magnetic force-assisted transfection using self-assembled complexes of nucleic acids and cationic lipids or polymers (nonviral gene vectors), which are associated with magnetic (nano) particles. These magnetic complexes are sedimented onto the surface of the cells to be transfected within minutes by the application of a magnetic gradient field. As the diffusion barrier to nucleic acid delivery is overcome, the full vector dose is targeted to the cell surface and transfection is synchronized. In this manner, the transfection process is accelerated and transfection efficiencies can be improved up to several 1,000-fold compared with transfections carried out with nonmagnetic gene vectors. This protocol describes how to accomplish the following stages: synthesis of magnetic nanoparticles for magnetofection; testing the association of DNA with the magnetic components of the transfection complex; preparation of magnetic lipoplexes and polyplexes; magnetofection; and data processing. The synthesis and characterization of magnetic nanoparticles can be accomplished within 3-5 d. Cell culture and transfection is then estimated to take 3 d. Transfected gene expression analysis, cell viability assays and calibration will probably take a few hours. This protocol can be used for cells that are difficult to transfect, such as primary cells, and may also be applied to viral nucleic acid delivery. With only minor alterations, this protocol can also be useful for magnetic cell labeling for cell tracking studies and, as it is, will be useful for screening vector compositions and novel magnetic nanoparticle preparations for optimized transfection efficiency in any cell type.     

综述——新型纳米生物材料的研发：磁性纳米材料：化学合成、功能化与生物医学应用

磁性纳米材料,由于其独特的磁学性能、小尺寸效应,被广泛应用于生物医学领域．本文总结了磁性纳米材料的化学设计与合成、表面功能化方法,及其在核磁共振成像、磁控治疗、磁热疗和生物分离等生物医学领域的应用进展．     

Fe3O4/ 生物可降解复合材料研究

磁性氧化铁纳米粒子因具有尺寸小、低毒性和超顺磁性等特点,已经引起了生物化工、医药工业领域的广泛关注。生物可降解高分子材料是生物医用高分子研究中最活跃的领域之一,已广泛用于外科手术缝合线,植入体材料及药物释放载体等。将Fe3O4和生物可降解高分子材料进行复合,可以扩大两者的应用范围,达到理想的治疗效果,并有望开创临床治疗的新时代。本文介绍了磁性四氧化三铁粒子的化学制备方法,包括共沉淀法、溶胶-凝胶法、微乳液法,并对各种方法的优缺点进行了比较;重点阐述了磁性壳聚糖,磁性聚乳酸,磁性PEG,磁性PCL复合材料的制备,及它们在酶的固定化、磁靶向药物及基因载体等医学领域的应用,显示了Fe3O4/生物可降解复合材料在医学领域的广阔应用前景;最后对复合材料走向临床应用所面临的问题及发展前景进行了讨论。     

Fc段受体结合蛋白(SPA-SPG)基因克隆表达及其在IgG纯化中的应用

利用生物信息学,遴选编码ＳＰＡ及ＳＰＧ蛋白的基因,进行密码子优化,将目的基因分割成互为重叠的小片段寡聚核苷酸链,采用一步升温后T4 DNA连接酶连接的方法,合成了编码ＳＰＡ及ＳＰＧ蛋白的融合基因.将其克隆到pSK质粒进行扩增, 经测序、修正后再克隆到表达载体,高效表达了带His6的融合蛋白——蛋白ＡＧ.将蛋白ＡＧ共价结合到表面带羧基的磁粒上,形成蛋白ＡＧ磁粒复合物,用此复合物可在1 h内从大鼠、小鼠、人、猕猴、马、羊及猪等常用实验动物血清中纯化ＩｇＧ.     

Insights into the mechanism of magnetofection using PEI-based magnetofectins for gene transfer

BACKGROUND: Gene delivery by the use of magnetic forces, so-called magnetofection, has been shown to enhance transfection efficiency of viral and non-viral systems up to several-hundred-fold. For this purpose gene carriers, such as polyethylenimine (PEI), are associated with superparamagnetic nanoparticles and complexed with plasmid DNA. Gene delivery is targeted by the application of a magnetic field. METHODS: To investigate the underlying mechanism, we studied the impact of the applied magnetic field on the transfection process of PEI-coated superparamagnetic iron oxide gene vectors (magnetofectins) using various cell lines. In particular, we addressed the question whether accelerated sedimentation of magnetofectins is the driving force or if the magnetic field itself directly influences the endocytic processing of the magnetofectins. The cellular uptake mechanism of magnetofectins was studied by electron microscopy and transfection experiments in the presence of various inhibitors that operate at different steps of endocytosis. RESULTS: In this study we could show that cellular uptake of magnetofectins proceeds obviously by endocytosis. Cellular uptake of magnetofectins behaves almost analogously as compared with PEI polyplexes. Besides unspecific endocytosis, apparently clathrin-dependent as well as caveolae-mediated endocytic uptake is involved. CONCLUSIONS: The magnetic field itself does not alter the uptake mechanism of magnetofectins. Obviously, the magnetic forces lead to an accelerated sedimentation of magnetofectins on the cell surface and do not directly affect the endocytic uptake mechanism. So further improvement of magnetic field application could lead to efficient targeting of gene expression into the desired organ and tissue in vivo.     

磁纳米识别探针的构造及其在真菌毒素检测中的应用进展

真菌毒素是一种由真菌产生的具有毒性的次级代谢产物,易引发严重的食品安全问题,不断探索更为高效准确的新型检测方法具有重要意义。磁纳米识别探针具有高效易分离、结合容量大、识别效果好、功能性强等优势,为复杂基质中痕量真菌毒素检测研究带来新方向。本文对磁纳米识别探针构造,由内向外对构成探针的磁纳米核心颗粒及其表面修饰物的种类及特点进行总结分析,在此基础上进而从探针的选择与功能、检测条件、检测灵敏度及特异性等方面,对近年来磁纳米识别探针在食品体系真菌毒素检测中的应用研究进行概述归纳,并对其未来的应用前景与发展方向进行展望。