一种体内高效表达反义RNA、三链形成RNA及Ribozyme的新载体

为了在体内高效表达反义RNA,三链形成RNA与Ribozxyme,通过PCR克隆技术构建了一种全新载体pBSKnerrU6’。该载体利用hsnRNA U6基因表达单元的特性,保留了U6RNA分子5’端维持分子稳定性的发夹和帽式结构的序列,缺失了其中间参与mRNA前体剪切的部分,取而代之的是3个限制性内切酶位点,Hela细胞核抽提物体外转录实验证明该载体可有效地表达U6变体RNA分子,该载体携带新霉素抗性基因neo^r,因而可用于真核细胞核细胞稳定转染的筛选。     

人类骨桥蛋白(hOPN)在细胞增殖中的功能研究

为了研究人类骨桥蛋白（hOPN)与293细胞增殖。细胞周期及与细胞周期有关基因表达的关系。并对其机制进行探讨。成功地构建了hOPN真核表达载体并获得了稳定表达hOPN的细胞系，也同时获得了稳定表达EGFP的细胞系，hOPN对293细胞具有促增殖效应，hOPN蛋白激活了293细胞细胞周期蛋白A的表达。实验结果表明：hOPN通过一定的信号通路刺激了细胞周期蛋白A的表达。加快了细胞进入通过S期，从而促进了细胞的增殖。     

藻蓝蛋白对Hela细胞CD59基因表达调控作用的研究

探讨了钝顶螺旋藻藻蓝蛋白(PC)对Hela细胞CD59基因表达的调控作用.以正常人CD59cDNA基因为模板,经PCR扩增后重组入真核表达质粒载体pALTER-MAX,然后利用阳离子脂质体(Lipfectamine-2000)将重组质粒和PcDNA共转染人子宫颈癌细胞(Hela)和对照用正常中国仓鼠卵巢细胞(Chinese hamster ovary,CHO)进行表达.用不同浓度的钝顶螺旋藻藻蓝蛋白作用于转染细胞,通过核酸分子杂交技术、免疫荧光标记法和ELISA法对细胞中CD59分子的表达进行检测.结果表明:成功构建了重组质粒pALTER-MAX-CD59,并将其导入真核细胞(Hela,CHO),经G418筛选获得了CD59分子高效表达的细胞克隆.用藻蓝蛋白作用于筛选出的转基因细胞,证实藻蓝蛋白可促进Hela细胞表面CD59蛋白的表达并抑制Hela细胞的增殖,而对于正常CHO细胞无明显作用.     

重组质粒pcFlaA在青石斑鱼肌肉中的表达研究

在哈维氏弧菌TS-628菌株鞭毛丝蛋白FlaA基因末端加上一段编码Flag短肽的核苷酸序列作为检测标记后,将该基因克隆到真核表达载体pcDNA3.1( ),酶切、PCR扩增及重组质粒测序证实基因片段插入正确,将该重组质粒命名为pcFlaA.将pcFlaA以肌肉注射方式免疫青石斑鱼.免疫后第7天开始检测鞭毛丝蛋白在石斑鱼肌肉中的表达状况,之后每隔1周检测1次,共检测4次.首先采用PCR技术在DNA水平检测重组质粒转染石斑鱼肌肉细胞的情况,再以RT-PCR法在mRNA水平上检测转染质粒在鱼肌肉中的转录,最后以免疫组化染色技术在蛋白质水平上检测目的蛋白的表达.结果在DNA及mRNA水平上均可检测到目的条带,在蛋白质水平上可检测到明显阳性位点,由此证实pcFlaA可以转染石斑鱼肌肉细胞并可在其中进行表达,而且质粒在鱼体内持续表达的时间至少1个月.     

牙鲆淋巴囊肿病毒主要衣壳蛋白基因片段真核表达载体的构建、表达与免疫效果评价

用淋巴囊肿病毒LCDV-cn感染牙鲆鳃细胞系FG-9307,提取细胞总RNA,用RTPCR法获得了LCDV-cn主要衣壳蛋白(MCP)0.6kb基因片段.将该LCDV-cn-MCP0.6kb片断克隆入真核表达载体pEGFP-N2,得到重组质粒pEGFP-N2-LCDV-cn-MCP 0.6.采用脂质体法将重组质粒转染入牙鲆鳃细胞系FEC,并进行瞬时表达.通过荧光显微镜观察和特异性RT-PCR检测,证实重组质粒pEGFP-N2-LCDV-cn-MCP 0.6kb已成功转染到FEC细胞,并得到了初步表达.将重组质粒肌注入牙鲆体内,检测牙鲆外周血、肠、脾脏、前肾和淋巴细胞的增殖反应、呼吸爆发活性及抗体产生水平.结果表明,构建的核酸疫苗pEGFP-N2-LCDV-cn-MCP 0.6可诱导牙鲆特异性体液免疫和细胞免疫,具有明显的免疫保护作用.     

小分子干扰RNA对斑马鱼VEGF基因的沉默作用

运用RNA干扰技术,针对斑马鱼血管内皮生长因子(VEGF)基因设计并合成了特异性小分子干扰RNA(siRNA),并从血管形成水平研究了siRNA对VEGF基因表达的影响。为了得到能在体内稳定表达的siRNA,进一步以pSilencer4.1-CMVVector为载体,合成了短发夹RNA(shRNA)表达模板并定向克隆到CMV启动子下游,构建了shRNA表达载体。定量碱性磷酸酶染色显示注射合成的siRNA后胚胎新血管生成(angiogenesis)为71.8%,NBT/BCIP血管染色显示表达载体可以引起斑马鱼胚胎肠下静脉、节间血管发育缺陷,原位杂交结果显示胚胎头部及前原肾管部位VEGF表达异常。     

抗RNA病毒相关生物材料

在介绍RNA病毒结构、生殖、复制和转录的基础上，综述了抗病毒策略，其中包括抗SARS药物设计，RNA干扰，DNA疫苗释放系统，调控蛋白与糖胺聚糖衍生物或类似物相互作用，天然药物及肺泡组织工程等。这些实例涵盖了RNA病毒与蛋白质、DNA及多糖等生物材料的相互作用。生物材料作为基质或载体正在向细胞或/和基因活化的第三代生物材料发展，可望在抗病毒中发挥作用。     

微小RNA纳米递送体系的构建及其研究进展

微小RNA是一种短链的、调节性非编码RNA,约由21～25个核苷酸组成,广泛存在动物、植物及病原微生物中,具有在翻译水平调控基因表达的功能,是一类重要的基因表达调节器。目前,已在人类基因组中鉴定出1 000多个miRNA,超过30%的基因受到miRNA调节。miRNA广泛参与细胞生长、分化及凋亡过程。研究表明,生物体内miRNA表达水平与神经障碍、心血管疾病、癌症和病毒感染等疾病密切相关。因此,miRNA已被广泛用作多种疾病的早期诊断、预后评估以及治疗靶点。利用RNA干扰(RNA interference)技术调控生物体内miRNA的含量已经引起越来越多的关注,通过向细胞内递送miRNA或者"反义"核苷酸可以实现对目标miRNA表达水平的调控,从而实现对基因失调所引起的疾病的治疗。目前,基于miRNA的治疗方法在癌症、新发传染病和其他疾病中表现出极大的潜力。然而,核苷酸链自身的特性,如负电性、易降解,导致其很难跨越细胞膜进入细胞。因此,合理设计纳米递送载体,对提高治疗基因的运载和治疗效率具有重要的意义。目前,随着新型材料的深入研究,已经报道有多种材料可以作为基因载体并用于细胞内递送miRNA。例如脂质体胶囊,它是利用磷脂双分子层膜所形成的囊泡包裹药物分子而形成的制剂,具有良好的生物相容性和稳定性,可以通过细胞内吞作用进入细胞内,从而实现对基因的递送。此外,利用不同材料的物理、化学特性,多种形式的纳米体系,如聚合物纳米颗粒、有机和无机纳米颗粒等已被开发设计成功能性纳米载体,可实现基因药物的靶向递送和智能刺激释放。本文将讨论细胞内miRNA的功能以及调控miRNA含量的方法,归纳纳米体系在递送miRNA治疗基因方面的研究进展,分析纳米递送体系的设计思路、方法以及作用机制。此外,还将根据目前的研究进展,讨论基于miRNA的疗法在新兴领域中的发展方向,以期为制备新型智能的基因递送体系提供参考。     

人膜补体调节蛋白MCP、CD59在稳定转染的细胞中的共表达及功能研究

采取内核糖体进入位点(IRES)策略构建含人的膜补体调节蛋白基因MCP和CD59的cDNA的双顺反子真核表达载体pcDNA3-MCPIRESCD59,以磷酸钙沉淀法转染NIH3T3细胞,用 G418筛选阳性克隆,并研究MCP和CD59双基因在稳定细胞系中的共表达及保护功能.PCR实验结果显示双基因稳定整合在异源细胞NIH3T3的染色体上,RT-PCR及Western印迹实验分别从 RNA水平和蛋白质水平证实了人补体调节蛋白分子MCP和CD59在细胞系中皆获得同步表达.检测连续传代30次的NIH3T3 pcDNA3-MCPIRESCD59,结果表明人MCP和CD59基因仍稳定整合在细胞基因组中,并未随着传代而丢失,为稳定的转双基因细胞系.补体依赖的细胞毒反应表明,pcDNA3-MCPIRESCD59转染细胞由于MCP和CD59的共表达获得了高于MCP或CD59单一表达时所提供的保护功效,能更好地抑制人补体依赖的细胞毒作用的发生,保护宿主细胞免受人补体的攻击.以上结果表明,所构建的双基因重组表达载体实现了不同人补体调节蛋白基因高效转移和高水平共表达,在克服超急性排斥反应的基因治疗中有潜在的应用价值.     

Stabilizing RNA by the sonochemical formation of RNA nanospheres

Biological macromolecules, including DNA, RNA, and proteins, have intrinsic features that make them potential building blocks for the bottom-up fabrication of nanodevices. Unlike DNA, RNA is a more versatile molecule whose range in the cell is from 21 to thousands of nucleotides and is usually folded into stem and loop structures. RNA is unique in nanoscale fabrication due to its diversity in size, function, and structure. Because gene expression analysis is becoming a clinical reality and there is a need to collect RNA in minute amounts from clinical samples, keeping the RNA intact is a growing challenge. RNA samples are notoriously difficult to handle because of their highly labile nature and tendency to degrade even under controlled RNase-free conditions and maintenance in the cold. Silencing the RNA that induces the RNA interference is viewed as the next generation of therapeutics. The stabilization and delivery of RNA to cells are the major concerns in making siRNAs usable drugs. For the first time, ultrasonic waves are shown to convert native RNA molecules to RNA nanospheres. The creation of the nanobubbles is performed by a one-step reaction. The RNA nanospheres are stable at room temperature for at least one month. Additionally, the nanospheres can be inserted into mammalian cancer cells (U2OS). This research achieves: 1) a solution to RNA storage; and 2) a way to convert RNA molecules to RNA particles. RNA nanosphere formation is a reversible process, and by using denaturing conditions, the RNA can be refolded into intact molecules.     

Modelling the dynamics of viral suppressors of RNA silencing

Virus infection in plants is limited by RNA silencing. In turn, viruses can counter RNA silencing with silencing suppressors. Viral suppressors of RNA silencing have been shown to play a role in symptom development in plants. We here study four different strategies employed by silencing suppressors: small interfering RNA (siRNA) binding, double-strand RNA (dsRNA) binding and degrading or inactivating Argonaute. We study the effect of the suppressors on viral accumulation within the cell as well as its spread on a tissue with mathematical and computational models. We find that suppressors which target Argonaute are very effective in a single cell, but that targeting dsRNA or siRNA is much more effective at the tissue level. Although targeting Argonaute can be beneficial for viral spread, it can also cause hindrance in some cases owing to raised levels of siRNAs that can spread to other cells.     

Quantum dot conjugates for targeted silencing of bcr/abl gene by RNA interference in human myelogenous leukemia K562 cells

Quantum dots (QDs) have been receiving a lot of attention recently for their unique fluorescence properties that can be used in drug discovery and bioimaging applications. We have in this article focused particularly on QDs and used it as a transfection vector as well as a fluorescence label for the RNA interference research. The siRNAs were designed to knock down the bcr/abl oncogene in leukaemia K562 cells. EDAC used as a cross-linker, COOH-functionalized QDs were conjugated with NH2-modified siRNAs to generate QD-siRNA conjugates. We also demonstrated their application to the K562 cells. Using such constructs, the delivery and transfection of siRNAs could be monitored by the presence of fluorescent QDs in the conjugates. QDs not only exhibited superior photostability for labeling cells but also worked as a good vector that remarkably increased the transfection efficiency of siRNAs into the cells. Cell proliferation was examined by the MTT assay and cell apoptosis by FACS. Our data have shown that the QD-siRNA conjugates could efficiently inhibit the viability of K562 cells and induced their apoptosis. In summary, QDs can be considered strong tools for the functional analysis of RNAi.     

Nanochannel Electro-Injection as a Versatile Platform for Efficient RNA/DNA Programming on Dendritic Cells

The utilization of dendritic cell (DC) vaccines is a promising approach in cancer immunotherapy, and the modification of DCs for the expression of tumor-associated antigens is critical for successful cancer immunotherapy. A safe and efficient method for delivering DNA/RNA into DCs without inducing maturation is beneficial to achieve successful DC transformation for cell vaccine applications, yet remains challenging. This work presents a nanochannel electro-injection (NEI) system for the safe and efficient delivery of a variety of nucleic acid molecules into DCs. The device is based on track-etched nanochannel membrane as key components, where the nano-sized channels localize the electric field on the cell membrane, enabling lower voltage (<30 V) for cell electroporation. The pulse conditions of NEI are examined so that the transfection efficiency (>70%) and biosafety (viability >85%) on delivering fluorescent dyes, plasmid DNA, messenger RNA, and circular RNA (circRNA) into DC2.4 are optimized. Primary mouse bone marrow DC can also be transfected with circRNA with 68.3% efficiency, but without remarkably affecting cellular viability or inducing DC maturation. These results suggest that NEI can be a safe and efficient transfection platform for in vitro transformation of DCs and possesses a promising potential for developing DC vaccines against cancer.     

Synthesis of stable silica-dye hybrid nanomaterial as DNA carrier

A new method is proposed for the fabrication of fluorescence-labeled and amine-modified silica nanoparticles for application as nonviral vectors in gene delivery. Highly monodisperse, stable fluorescent silica nanoparticles were prepared using 2,5-bis(5-tert-butyl-2-benzoxazolyl)thiophene and the water-in-oil microemulsion method. The green-fluorescent-protein gene can be easily combined onto the positively charged surfaces of nanoparticles to form a nanoparticle-DNA complex. The nanoparticle-DNA complex successfully passed through various barriers into the HeLa and HEK 293 K cells. The cytotoxicity of the PEI-coated and BBOT-encapsulated silica nanoparticles on both the HeLa and HEK 293T cell lines was found to be at an acceptable level for use as gene carriers when the particle concentration was below 125 microg/ml. The fluorescence intracellular images confirm the successful delivery of the nanoparticle-DNA complex and gene expression. The present work suggests the potential use of dye-incorporated silica nanoparticles in nonviral gene delivery.     

Design of novel polysaccharidic nanostructures for gene delivery

The goal of the present work was to develop a new synthetic nanosystem for gene delivery. For this purpose, we chose two polysaccharides, hyaluronic acid (HA) and chitosan (CS), as the main components of the nanocarrier. Nanoparticles with different hyaluronate:chitosan (HA:CS) mass ratios (0.5:1 and 1:1) and different polymer molecular weights (hyaluronate 170 (HA) or <10?kDa (HAO) and chitosan 125 (CS) or 10-12?(CSO)?kDa) could be obtained using an ionic crosslinking method. These nanoparticles were loaded with pDNA and characterized for their size, zeta potential and pDNA association efficiency. Moreover, their toxicity and ability to transfect the model plasmid pEGFP-C1 were evaluated in the cell line HEK 293, as well as their intracellular fate. The results showed that HA:CS nanoparticles have a small size in the range of 110-230?nm, a positive zeta potential of +10 to +32?mV and a very high pDNA association efficiency of 87-99% (w/w). On the other hand, nanoparticles exhibited low cell toxicity and transfection levels up to 25% GFP expressing HEK?293 cells, lasting for the whole observation period of 10 days. We also provide basic information about the role of both polymers, HA and CS, and the effect of their molecular weight on the effectiveness of the resulting DNA nanocarrier, being the highest transfection levels observed with HAO:CSO 1:1 nanoparticles. In?conclusion, HA:CS nanoparticles are promising carriers for gene delivery.     

Protamine and chloroquine enhance gene delivery and expression mediated by RNA-wrapped single walled carbon nanotubes

The use of non-viral vectors as delivery systems in gene therapy has been extensively studied recently owing to their advantages over viral vectors. Here, we propose a new gene delivery system based on the use of RNA-wrapped single-walled carbon nanotubes (SWCNTs) complexed with the cationic protein, protamine and the drug chloroquine. Protamine was selected as a cationic protein acting as bridge between negatively charged RNA-wrapped SWCNTs and plasmid DNA. Protamine also contains a nuclear localization signal which enhances the expression of the transfected gene. The drug chloroquine, a lysosomotropic compound which has been reported to increase the transfection efficiency, was attached to RNA-wrapped SWNTs by ionic interactions. The simultaneous delivery of the drug chloroquine with plasmid DNA clearly showed an enhanced gene delivery and expression. The levels of gene expression were quantified using the luciferase reporter gene as model. Optimal conditions for transfection and gene expression were obtained and cytoxicity of the carbon nanotube complexes measured. The optimal complexes were shown to efficiently deliver plasmid DNA for efficient gene expression and may thereby be useful as gene delivery systems for gene therapy.     

Cholesterol tethered bioresponsive polycation as a candidate for gene delivery

The efficient unpacking of viral protein shell gave the inspiration for the synthesized vectors. In this research, novel cholesterol tethered bioresponsive polyethylenimine (PEI) was specially designed via disulfide-containing cross-linker. The cholesterol lipid had proved to increase the permeability of gene vector through cell membrane. The acid–base titration indicated that the synthesized polycation possessed efficient proton sponge effect, which was suggested to increase endosomal release of pDNA complexes into the cytoplasm. The cholesterol tethered polycation could effectively induce DNA condensation and form spherical particles with diameter about 200 nm at N/P ratio of 10. At glutathione concentration of 3 mM, the polyplexes were unpacked due to the bioresponsive cleavage of the disulfide bonds. The in-vitro experiment indicated that the polyplexes showed efficient transfection efficiency to HEK293T cells. All the results indicated that the bioresponsive polycation could be served as an effective trigger to control the release of DNA at the intracellular environment. The novel bioresponsive polycation might have great potential in non-viral gene delivery research and application.     

Evaluation of androgenic activity of nutraceutical-derived steroids using mammalian and yeast in vitro androgen bioassays

Androgenic steroids marketed online as nutraceuticals are a growing concern in sport doping. The inability of conventional mass spectrometry (MS)-based techniques to detect structurally novel androgens has led to the development of in vitro androgen bioassays to identify such designer androgens by their bioactivity. The objective of this study was to determine the androgenic bioactivity of novel steroidal compounds isolated from nutraceuticals using both yeast and mammalian cell-based androgen bioassays. We developed two new in vitro androgen bioassays by stably transfecting HEK293 and HuH7 cells with the human androgen receptor (hAR) expression plasmid together with a novel reporter gene vector (enhancer/ARE/SEAP). The yeast β-galactosidase androgen bioassay was used for comparison. Our new bioassay featuring the enhancer/ARE/SEAP construct (-S) displayed simpler assay format and higher specificity with lower sensitivity compared with the commonly used mouse mammary tumour virus (MMTV)-luciferase. The relative potencies (RP), defined as [EC(50)] of testosterone/[EC(50)] of steroid, of nutraceutical extracts in the yeast, HEK293-S, and HuH7-S, were 34, 333, and 80,000 for Hemapolin; 208, 250, and 80 for Furazadrol; 0.38, 10, and 106 for Oxyguno; 2.7, 0.28, and 15 for Trena; and 4.5, 0.1, and 0.4 for Formadrol, respectively. The wide discrepancies in rank RP of these compounds was reconciled into a consistent potency ranking when the cells were treated with meclofenamic acid, a nonselective inhibitor of steroid metabolizing enzymes. These findings indicate that steroids extracted from nutraceuticals can be converted in vitro into more or less potent androgens in mammalian but not in yeast cells. We conclude that the putative androgenic bioactivity of a new compound may depend on the bioassay cellular format and that mammalian cell bioassays may have an added benefit in screening for proandrogens but sacrifice specificity for sensitivity in quantitation.