程序组装多功能化纳米基因载体

基因治疗成功的关键之一是采用安全高效的载体递送基因。多功能化的非病毒基因载体可克服转染过程中的多种屏障,提高转染效率。通过科研实践和文献查阅,本文总结出实现载体多功能化的三种程序组装方式,即层层自组装、共聚物自组装和脂质掺入,并对近年来国内外通过程序组装构建多功能非病毒基因载体的研究进展做一简要综述。     

用作基因传递系统的阳离子纳米载体的 细胞毒性及其机制研究进展

作为基因治疗中的非病毒基因载体,阳离子纳米载体可通过电荷作用与核酸类药物相结合,具有广阔的应用前景。然而,其细胞毒 性,主要表现为诱导细胞凋亡,限制了其临床开发与应用,也成为阳离子纳米载体研究所关注的重点。揭示和准确评价阳离子纳米载体的 细胞毒性及其机制,将有助于设计和开发更安全、更高效地用于基因传递的阳离子纳米载体。综述常用作基因传递系统的阳离子纳米载体 材料阳离子脂质体、聚乙烯亚胺、多聚赖氨酸、聚苯乙烯纳米粒以及其他阳离子聚合物的细胞毒性及其机制研究进展。     

载基因壳聚糖纳米粒的制备及免疫增强作用的初步研究

摘 要 目的: 制备壳聚糖载基因纳米粒,并对其体外转染效率及其在小鼠体内的免疫增强效果进行初步研究。方法: 以本课题组构建的口蹄疫DNA疫苗为模型药物,采用复凝聚法制备纳米粒;用透射电镜观察形态;用纳米粒度分析仪测定粒径、多分散度和zeta电位;凝胶阻滞分析测定基因在纳米粒中的位置;用体外基因转染实验评价纳米粒的转染活性。用载基因壳聚糖纳米粒免疫雌性Balb/c小鼠,检测免疫小鼠的细胞免疫和体液免疫水平。结果: 所制备的载基因纳米粒形态规则、大多成球形,平均粒径约为150nm,多分散度＜0.26,zeta电位约为21mV;凝胶分析结果表明质粒DNA与壳聚糖分子间可以通过电性结合作用而完全结合,基因几乎全部被包裹在纳米粒内部;体外基因转染实验表明壳聚糖作为一种新型的非病毒基因递送载体能够高效传递DNA进入BHK-21细胞,基因能够在该细胞中高效表达;小鼠免疫实验表明纳米粒不仅能诱导机体产生较高的细胞免疫水平,而且体液免疫水平也显著提高。结论: 壳聚糖纳米粒能将基因递送到细胞内并且能够表达,小鼠免疫实验显示其具有良好的免疫增强效果。     

纳米粒载体研究进展

目的:介绍纳米粒载体的制备、优点和应用进展,为纳米粒在新的领域的应用提供依据.方法:以纳米粒制备方法和基质材料的不断发展以及各个领域的应用为线索来综述.结果:纳米粒的制备方法有离子交换法、乳化法和自组装法,基质材料有蛋白质和多糖等,纳米粒本身无毒性,能增加所载物的溶解度和生物利用度,提高靶向性及效率,在抗肿瘤治疗、神经系统治疗和药物检测等多方面有广泛的应用.结论:纳米粒作为一种载体,在药物临床研究、药理研究和生物分析等方面都得到了广泛应用,并有可能开发为其他领域的运送体系,有广阔的应用前景.     

钛- 载药纳米复合材料的组装及其性质的研究

目的:构建一种能结合钛表面的载药纳米粒及钛-载药纳米复合材料的组装和性质研究。方法:(1)多巴胺修饰的非离子表面活性剂多巴胺-泊洛沙姆188(Dop-Poloxamer188)的合成和检测;(2)Dop-Poloxamer188作为表面活性剂、PLGA作为油相基质,制备纳米粒及纳米粒载药和表征;(3)钛片的预处理及钛片与修饰后的纳米粒的结合;(4)纳米粒修饰后的钛表面的表征。结果:新合成的Dop-Poloxamer188在285 nm左右有紫外吸收峰,说明多巴胺成功的修饰在Poloxamer188的两端;Dop-Poloxamer188能和PLGA制备出很好的纳米粒,平均粒径在110 nm左右,PDI小于0.1;多巴胺修饰的纳米粒与钛片通过简单的浸渍过程结合后,通过水接触角、场发射扫面电镜(Fe-SEM)、荧光显微镜、X射线光电子能谱(XPS)等仪器检测都显示多巴胺修饰的纳米粒成功且牢固的修饰在钛片表面。结论:成功达到钛表面的载药纳米粒修饰的目的,为钛种植体的载药系统提供了新的思路和方法。     

生物可降解聚合物纳米粒给药载体

生物可降解聚合物纳米粒用于给药载体具有广阔的前景。本文综述了生物可降解聚合物纳米粒给药载体领域的最新进展 :包括纳米粒表面修饰特性、药物释放、载多肽和蛋白质等生物大分子药物传输中的潜在应用。     

可降解的非病毒基因载体的研究进展

选择合适的基因载体是基因治疗成功与否的一个关键环节,合适的基因载体应该具备较高转染效率,较好的组织相容性以及生物安全性等特点。相较于传统的病毒基因载体,非病毒基因载体具有低免疫原性,易于制备以及载药量大等特点,是较为理想的基因载体。而寻求可降解的非病毒基因载体可使其更为安全深入的应用到体内以及临床,意义重大。本研究就对常见的几种可降解的基因载体的转染效果及相关改性做一综述,为研究可降解非病毒基因载体的新方法提供新思路。     

非病毒基因治疗的研究进展

非病毒基因治疗是相对于病毒性基因治疗而言,指采用非病毒的载体进行的基因治疗。非病毒的基因载体比病毒性基因载体具有高安全性、低免疫原性及易于生产的特点。本文就非病毒基因治疗所采用的主要方法、面蜂的主要问题及发展方向作一概括的介绍。随着人类对疾病发病分子机制的深入研究及人类基因组计划的实施,非病毒基因治疗将在人类疾病的治疗中发挥重要作用。     

纳米粒药物载体在抗癌领域的研究进展

抗癌药物载体是合成化学在药学领域的新发展,其中纳米粒药物载体材料以独特的结构特点、较大的载药量等优势受到国内外广泛关注。经亲水聚合物修饰后的纳米粒药物载体有较高的生物相溶性,且药物分子能靶向作用于癌细胞,延长药物半衰期,提高生物利用度,具有生物可降解性,消除毒副作用。在抗癌领域显示出研究意义和应用价值。本文就纳米粒药物载体研究进展进行综述述。     

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.     

Synthesis of aminated polysorbate 80 for polyplex‐mediated gene transfection

To develop novel gene delivery carriers, aminated polysorbate 80 (P80‐NH₂) was synthesized with strong positively charged properties through the introduction of three amine groups. The resulting P80‐NH₂ and DNA polyplex exhibited superb condensation abilities due to the high densities of positively charged amines groups. Size and surface charge of polyplex were shown to be well suited for cellular internalization. In addition, the P80‐NH₂/DNA polyplex demonstrated acceptable transfection efficiency in HeLa cells and was nontoxic relative to the conventional 25‐kDa polyethyleneimine system. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Successful transfection of hepatoma cells after encapsulation of plasmid DNA into negatively charged liposomes

The application of conventional cationic liposomes/DNA complexes in gene transfer was hampered due to their large size, instability, and limited transfection site in vivo. In this report, we described a dialysis-based method and produced small, stable, and negatively charged DNA-containing liposomes composed of low content of cationic lipid and high content of fusogenic lipid. The liposomes were relatively spherical with a condensed core inside, and exhibited small size with narrow particle size distribution. The encapsulation efficiency of the liposomes was 42.53 +/- 2.29%. They were stable and showed enough protective ability to plasmid DNA from degradation after incubation with different amounts of DNase. Twenty-fold higher transfection efficiency for the liposomes was achieved when compared with that of naked plasmid DNA and no toxicities to hepatocellular carcinoma cells were observed. Our results indicate that the negatively charged DNA-containing liposomes can facilitate gene transfer in cultured cells, and may alleviate the drawbacks of the conventional cationic liposomes/DNA complexes for gene delivery in vivo.     

Positively charged DNA-binding proteins cause apparent cell membrane translocation

Several positively charged DNA-binding proteins such as the human immunodeficiency virus Tat protein, the Antennapedia (Antp) homeobox protein, and the herpes simplex virus VP22 protein have been reported to translocate across cell membranes and accumulate in cell nuclei. The import occurs by a poorly understood mechanism that appears to be receptor- and energy-independent. We showed that both VP22 and the positively charged histone H1 adhered to the cell membrane of living cells and were not removed by extensive washing. However, after fixation the proteins relocated to the cell nucleus. The nuclear accumulation of VP22 and histone H1 after fixation shows that positively charged proteins may appear to translocate across the cell membrane because of a fixation artifact. The majority of studies on "membrane permeable" proteins and peptides have been performed using fixation techniques, and our study shows that influx of these proteins may occur during fixation rather than in living cells.     

Separating lysozyme from bacteriophage P22 in two-phase aqueous micellar systems

This communication demonstrates that two-phase aqueous mixed (nonionic/ionic) micellar systems have the potential for improving the separation of proteins from viruses. Specifically, two separation experiments were performed to show that the addition of the anionic surfactant sodium dodecyl sulfate (SDS) to the two-phase aqueous nonionic n-decyl tetra(ethylene oxide) (C(10)E(4)) micellar system increases the yield of a model net positively charged protein, lysozyme, in the micelle-rich phase from 75 to 95%, while still maintaining approximately the same yield of a model net negatively charged virus, bacteriophage P22, in the micelle-poor phase (97% vs. 98%).     

Sequence Analysis of the Streptococcus mutans Ingbritt dexA Gene Encoding Extracellular Dextranase

The complete nucleotide sequence (3,747 bp) of the dextranase gene (dexA) and flanking regions of the chromosome of Streptococcus mutans Ingbritt (serotype c) were determined. The open reading frame for dexA was 2,550 bp, ending with a stop codon TGA. A putative ribosome-binding site, promoter preceding the start codon, and potential stem-loop structure were identified. The presumed dextranase protein (DexA) consisting of 850 amino acids was estimated to have a molecular size of 94,536 Da and a pI of 4.79. The nucleotide sequence and the deduced amino acid sequences of S. mutans dexA exhibited homologies of 57.8% and 47.0%, respectively, to those of Streptococcus sobrinus dex. The homologous region of dex of S. sobrinus was in the N-terminal half. The C terminus of DexA consisted of a hexapeptide LPQTGD, followed by 7 charged amino acids, 21 amino acids with a strongly hydrophobic character, and a charged hexapeptide tail, which have been reported as a common structure of C termini of not only the surface-associated proteins of Gram-positive cocci but also the extracellular enzymes such as β-fructosidase of S. mutans and dextranase of S. sobrinus. The DexA protein had no significant homology with the glucosyltransferases, the glucan-binding protein, or the dextranase inhibitor of mutans streptococci.     

A Three-protein Charge Zipper Stabilizes a Complex Modulating Bacterial Gene Silencing

The Hha/YmoA nucleoid-associated proteins help selectively silence horizontally acquired genetic material, including pathogenicity and antibiotic resistance genes and their maintenance in the absence of selective pressure. Members of the Hha family contribute to gene silencing by binding to the N-terminal dimerization domain of H-NS and modifying its selectivity. Hha-like proteins and the H-NS N-terminal domain are unusually rich in charged residues, and their interaction is mostly electrostatic-driven but, nonetheless, highly selective. The NMR-based structural model of the complex between Hha/YmoA and the H-NS N-terminal dimerization domain reveals that the origin of the selectivity is the formation of a three-protein charge zipper with interdigitated complementary charged residues from Hha and the two units of the H-NS dimer. The free form of YmoA shows collective microsecond-millisecond dynamics that can by measured by NMR relaxation dispersion experiments and shows a linear dependence with the salt concentration. The number of residues sensing the collective dynamics and the population of the minor form increased in the presence of H-NS. Additionally, a single residue mutation in YmoA (D43N) abolished H-NS binding and the dynamics of the apo-form, suggesting the dynamics and binding are functionally related.     

Local gene silencing in plants via synthetic dsRNA and carrier peptide

Quick and facile transient RNA interference (RNAi) is one of the most valuable plant biotechnologies for analysing plant gene functions. To establish a novel double‐strand RNA (dsRNA) delivery system for plants, we developed an ionic complex of synthetic dsRNA with a carrier peptide in which a cell‐penetrating peptide is fused with a polycation sequence as a gene carrier. The dsRNA–peptide complex is 100–300 nm in diameter and positively charged. Infiltration of the complex into intact leaf cells of Arabidopsis thaliana successfully induced rapid and efficient down‐regulation of exogenous and endogenous genes such as yellow fluorescent protein and chalcone synthase. The present method realizes quick and local gene silencing in specific tissues and/or organs in plants.     

Molecular cloning of a light-inducible gene (lipA) encoding a novel pilin from Arthrobacter photogonimos

Development of pili on cells of Arthrobacter photogonimos is induced by photo-oxidative conditions. The nucleotide sequence was determined of a light-inducible gene (lipA) that encodes the precursor of a light-inducible pilin (designated LIP), a polypeptide of 212 amino acids. The N-terminal leader peptide includes a typical signal sequence with a consensus cleavage site for signal peptidase I after residue 28, which should generate N-terminal arginine. However, the next amino acid, alanine, is the N-terminal residue of the mature protein. The abundance of charged amino acids (27% of total), a calculated pI of 9.98, and recovery of mostly monomers when cells were washed with 1 M NaCl suggest that electrostatic interactions play a dominant role in association of LIP, a novel mechanism for assembly of pili.     

The fdp1 and cif1 mutations are caused by different single nucleotide changes in the yeast CIF1 gene

Abstract The allelism between the mutations cif1 and fdp1 from Saccharomyces cerevisiae has been demonstrated using PCR techniques and complementation of function. The cif1 mutation results in a shortened version of the protein while the fdp1 mutation introduces a charged residue in a highly hydrophobic stretch.