【作者】 朱诗国；

【导师】 李桂源；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2003， 博士

【摘要】 无论是在基因表达、功能与调节等基础研究方面，还是在疾病的治疗、预防，如基因治疗、DNA疫苗等临床应用方面，DNA传递始终是一个强有力的普遍应用工具，而且也是其中的核心技术。DNA传递的效率直接影响到其基础研究与临床应用的成效。传统的DNA传递系统分为病毒载体介导系统和非病毒载体介导系统。病毒载体介导系统是迄今为止最有效的DNA传递工具，其传递效率通常高达90％以上，最近临床基因治疗中75％应用病毒载体介导系统。尽管如此，由于该系统存在的自身局限性，如病毒毒性与免疫原性，有限的DNA装载量，载体组装难度大、花费高等，其应用受到很大限制。非病毒载体介导系统虽无病毒毒性和免疫原性等缺陷，但其传递效率一直不如病毒载体介导系统，如何提高非病毒载体介导系统的传递效率于是成为瓶颈问题。近来，随着纳米生物技术的飞跃发展，以纳米颗粒为基础的非病毒纳米基因传递载体系统的出现有望突破这一瓶颈，实现安全、高效的DNA传递，为基因表达与功能研究及其临床应用创造最佳条件，提供最好工具。 在先前的研究中，Luo D & Saltzman WM使用生物相容性的硅纳米颗粒进行基因转移，尽管它们自身不能传递DNA，但它们可以通过增加DNA—转染试剂复合物到达细胞表面的物理浓度而使最好的商品化转染试剂的基因转染效率增加8.5倍以上。这一研究结果提示，硅纳米颗粒如果用适当的阳离子聚合物，如多聚赖氨酸，改性修饰之朱诗国博士学位论文 新型纳米基因转运体与新型主物荧光纳米颗粒的矾’制后有可能获得有效的质粒DNA和反义ODN传递能力。 为此，本研究首先运用OP10／环己烷／氨水微乳液体系，以正硅酸乙酯为原料，成功研制了一批不同粒径的硅纳米颗粒，并通过正交试验分析了该体系中各组份对硅纳米颗粒尺寸的影响。结果表明在OPJ／环己烷／氨水微乳液体系中，水与 OP、 的摩尔比以及水与 TEOS的摩尔在控制 S i NP尺寸方面起抉定性作用，其次为氨水浓度，且随着其摩尔比的减小和氨水浓度的下降 SmP的粒径趋于最小。DNA结合滞留试验表明，在正常生理条件下 S i NP不能结合质粒 DNA。而随后的修饰研究发现，小粒径的硅纳米颗粒（约20 urn）表面羟基被活化后，它可与多聚赖氨酸结合形成形态规则、分布均匀的PMSWP，而大粒径的硅纳米颗粒则不能被修饰或修饰效果较差。这提示多聚赖氨酸对硅纳米颗粒的修饰与颗粒表面效能和Zeta电位密切相关，这种直接利用颗粒的表面效能和表面电位进行纳米颗粒改性修饰的方法，避免了复杂的化学反应和有毒化学物质的涉入，是一种新型的纳米颗粒改性修饰方法。进一步的细胞毒性研究表明，我们所制备的PMS十P细胞毒性极低，可能是一种良好的新型基因传递载体。 继而根据正交分析结果，按照最佳的合成方案合成了一批粒径小，分布更窄，形态更规则的硅纳米颗粒。在此基础上利用PLL进行改性修饰，制备出良好修饰的PMS—NP。DNA凝胶滞留研究和沉淀试验表明，随着PMS—NP与质粒DNA结合比例的增加，可逐渐出现的DNA滞留条带，上清液中 DNA浓度逐渐减少；达到 3 0：1后MA被完全滞留，上清液中检测不到质粒DNA。而且 PMS—NP与质粒DNA的 12朱诗国博十学位论义 新型纳爪基旧转运休与新型生物荧光纳米顺粒的汕划结合在40 Inin后达到平衡。进一步的DNasel消化试验和细胞转染研究发现，PMS—NP与质粒DNA结合后不仅能有效的保护DNA不被核酶降解，而且能有效的将质粒DNA转移到细胞内，产生高水平的报道基因表达。这些研究结果提示，PMS—NP是一新型的非病毒纳米颗粒基因转移载体，在基因结构与功能研究、基因治疗等领域具有广阔的应用前景。 反义ILNA是与特异基因互补配对的单链合成DNA序列。它通过Watson（rick硷基互补配对与靶基因的前mRNA或mRNA杂合，这种杂合既可直接阻断mMA的翻译，又可以RNA－DNA杂交复合物的形式激活 RNase H介导的 RNA特异性降解，从而在 mRNA和蛋白水平抑制基因表达。反义RNA作为肿瘤和感染性疾病的潜在治疗试剂己经在世界上众多实验室得到了验证，因而在感染性疾病和恶性肿瘤基因治疗中具有重要的战略意义。但是，为使反义技术广泛应用，有一个关键性的问题—一极低的的细胞渗透能力，不得不很好的解决。为增加 ODN的细胞内传递，避兔非特异性反应，颗粒载体，如脂质体或纳米颗粒的应用可能是解决ODN传递问题的最好方法。在药物载体中，生物可降解的或不可降解的纳米颗粒以一种有效的行为显示出结合和传递ODN的巨大潜能。甚至在某些‘倩况下，由于纳米颗粒良好的稳定性，它们显得比脂质体更为合适。 既然PMS—NP能够传递质粒DNA，自然使人想到它或许也是良好的反义传递载体。为此，本研究通过反义ODN结合和血浆蛋白切割实验研究其反义0＊N结合和保护能力：然后利用＊＊C标记的0＊N， 13朱更多还原

【Abstract】 DNA delivery has become a powerful popular research tool for elucidating gene structure, regulation, and function, as well as treating and controlling diseases, such as gene therapy and DNA vaccination. The efficiency of DNA delivery directly affects its application in basic research and clinical setting. Traditionally, DNA delivery systems have been classified as viral vector-mediated systems and nonviral vector-mediated systems. Viral systems are by far the most effective means of DNA delivery, achieving high efficiencies (usually > 90%) for both delivery and expression. In fact, around 75% of recent clinical protocols involving gene therapy use recombinant viral-based vectors for DNA delivery. As yet, however no definitive evidence has been presented for the clinical effectiveness of any gene therapy protocol. The impotence of current methodology is attributable to the limitation of viral-mediated delivery, including toxicity, restricted targeting of specific cell line, limited DNA carrying capacity, production and packaging problems, recombination, and high cost. Furthermore, the toxicity and immunogenicity of viral systems also hamper their routine use. For these reasons, nonviral systems have become increasingly desirable in basic research and clinical settings. But nonviral delivery efficiency is always less than viral systems. With the rapid development of nanobiotechnology, nanoparticles-basednonviral-mediated systems will help, to alleviate the gene delivery bottle-neck, and achieve the ability to safe, efficient, and targeted DNA delivery, and provide the best means for gene structure,, function research, as well as clinical settings.In the previous study, Luo D & Saltzman WM used biocompatible silica nanoparticles, which by themselves do not deliver DNA, to concentrate DNA-transfection reagent complexes at the surface of cell monolayers and enhanced transfection efficiency by up to 8.5-fold over the best commercially available transfection reagents. Their results suggested if silica nanoparticles were modified with polycations, such as poly-1-lysine, they maybe achieve the ability to efficient deliver DNA and antisense ODN.In the current research, silica nanoparticles (SiNP) were synthesized firstly in a microemulsion system polyoxyethylene nonylphenyl ether (OP-10)/ cyclohexane/ ammonium hydroxide, at the same time the effects of SiNP size and its distribution were elucidated by orthogonal analysis; then poly-1-lysine (PLL) was linked on the surface of SiNP by nanoparticle surface energy and electrostatically binding; lastly a novel complex nanomaterial-poly-1-lysine-midified silica nanoparticles (PMS-NP) was prepared. The analysis of plasmid DNA binding and DNase I enzymatic degradation discovered that PMS-NP could bind DNA, and protect it against enzymatic degradation. Cell transfection showed PMS-NP couldefficiently transfer pEGFP-C2 or pSV-p-gal plasmid DNA into HNE1 or Hela cell line. These results indicated PMS-NP is a novel nanoparticulate carrier for plasmid DNA delivery, and would probably play an important role in gene structure and function research as well as gene therapy.Antisense oligonucleotides are single-stranded synthetic DNA sequences designed to be complementary to a specific gene. Hybridization with target pro-mRNA or mRNA through Watson-Crick base-pairing can block sterically the translation of this transcript, or activate RNase H-mediated degradation of the RNA strand in RNA-DNA heteroduplexes and subsequently inhibit gene expression at both mRNA and protein levels. Antisense as a potential therapeutic agent against neoplastic and infectious diseases has been tested in numerous research laboratories around the world. However, one of the crucial problems to broad application of antisense technology, the low intracellular penetration, has to be solved. In order to increase ODN intracellular delivery, to avoid non-specific aptameric effects, the use of particulate carriers such as liposomes or nanoparticles, may be considered as being the more realistic更多还原