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。     

短发夹RNA介导RNA干涉的实验学控制研究

采用载体介导的RNA干涉(RNA interference,RNAi)技术抑制HEK293H细胞中外源报告基因的表达,并设置一系列的实验学控制以探讨其在RN加应用研究中的价值及意义。运用pAVU6 27,构建了针对增强型绿色荧光蛋白(EGFP)基因的一系列短发夹RNA表达载体,并用脂质体法转染于．HEK293H细胞中,检测EGFPmRNA及蛋白表达水平。瞬时转染48h后,RT-PCR及Western blot结果表明,针对EGFP三个靶点的干涉质粒均不同程度地抑制细胞内EGFP的表达,但仅转录正反义链、链内1—2个碱基错配的干涉载体不能介导特异的RNAi效应。此外,共转染实验结果表明干涉质粒两两间无相互增强或抑制的RNAi效应。在此基础上,通过G418筛选生成了HEK293H EGFP稳定表达株,在转染后不同时间点检测EGFPmRNA及蛋白表达变化,发现RNAi效应在一定时间范围内呈现明显的时间依赖性。这些结果表明,有效的实验控制在RNAi研究中十分必要,具有重要的参考价值。基于载体表达的RNAi作用呈现明显的时间依赖性效应,为RNAi应用研究提供了一定的参考及借鉴价值。     

大小家鼠4．5S RNA基因的克隆与序列比较

4．5SRNA基因序列是较晚发现的一种逆转座子,起源于7SLRNA。其转录物4．5SRNA存在于核内基质中,是由RNA聚合酶III转录的一类小RNA分子,在分类上属于核小RNA分子（snRNA）,本文利用设计的特异引物,从两种鼠类的总DNA中利用PCR技术扩增出4．5SRNA的基因,并将其连接到pGEM3Zf（＋）质粒载体上,然后转化大肠杆菌克隆,进行测序,并与已报道的三种鼠类的4．5SRNA的基因序列进行比较,对该基因的系统进化进行初步研究。     

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。     

淋巴囊肿病病毒主要衣壳蛋白基因片段原核载体的构建及表达

淋巴囊肿病病毒感染牙鲆鳃细胞系FG-9307，提取细胞总RNA，采用RT-PCR法成功获得了淋巴囊肿病病毒主要衣壳蛋白0．6kb基因片段。构建其原核表达载体，IPTG诱导表达。结果表明，该融合蛋白分子量约48kD，可与抗LCDV多克隆血清特异反应。为LCDV基因工程疫苗的研制奠定了实验基础。     

胶原纤维固化单宁对U6+的吸附特性研究

采用胶原纤维固化单宁(CFIT)作为吸附材料,系统研究了该吸附剂在模拟放射性废水中对U6+的吸附特性,离子溶液的pH值、吸附剂量、温度、时间和初始浓度等因素对U6+去除率的影响都进行了初步探讨,对吸附过程中的反应动力学、热力学参数以及等温吸附规律进行了分析。胶原纤维固化单宁对U6+有良好的吸附效果,当pH值在5.5左右,吸附时间为4h时,铀的去除率可达96.58%。通过吸附热的计算得出胶原纤维固化单宁在高温下的吸附可自发进行(ΔH=19.2942kJ/mol)。等温吸附研究表明,静态吸附动力学可以用准二级速度方程来描述,胶原纤维固化单宁对U6+的吸附行为符合Langmuir等温吸附方程,说明胶原纤维固化单宁对U6+的吸附是以单分子层吸附(化学吸附)为主,通过拟合得出最大吸附量为352.1127mg/g。     

RNA分子能自我组装成生物支架

美国研究人员表示,他们找到了一种方法,可以让纳米大小、能够对抗疾病的RNA（核糖核酸）分子自我组装成具有治疗效果的生物支架。得到的支架除了具有很好的功能扩展性，还可用作“桥梁”．向人体递送抗病药物。该研究成果将发表在最新一期《自然·纳米技术》杂志上。     

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。     

计算机在流感病毒(Influenza RNA)结构分析领域的应用

甲型H1N1流感病毒(influenzavirusA)基因组含有8个RNA基因片段,本文主要介绍了RNA的结构,以及目前计算机在RNA结构分析领域的应用现状,以及应用最多的预测工具。     

牙鲆生长激素基因的克隆及其在大肠杆菌中的融合表达

采用逆转录聚合酶链式反应(RT-PCR)方法,从牙鲆(Paralichthys olivaceus)脑垂体总RNA中扩增出编码牙鲆生长素(GH)成熟肽基因序列。重组至融合表达载体pGEX-4T-3中,构建成牙鲆GH基因融合表达载体pGEX-gh,转化大肠杆菌BL21(DF3),筛选阳性克隆,IVIG诱导表达。经SDS-PAGE电泳显示在45kD处有特异的蛋白条带出现,该蛋白的表达量随诱导时间的延长而增加,3h达最高值,达到细胞总蛋白的18．3％。该融合蛋白在胞内主要以包涵体状态存在,经优化表达条件,成功地获得了可溶性的融合蛋白,经Glutathione Sepharase 4B凝胶纯化后用Western-blotting检测表明其为牙鲆生长激素,并通过酶联免疫吸附受体法证实其具有生物学活性。     

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.     

The passage of homopolymeric RNA through small solid-state nanopores

Solid-state nanopores are widely acknowledged as tools with which to study local structure in biological molecules. Individual molecules are forced through a nanopore, causing a characteristic change in an ionic current that depends on the molecules' local diameter and charge distribution. Here, the translocation measurements of long (~5-30 kilobases) single-stranded poly(U) and poly(A) molecules through nanopores ranging from 1.5 to 8 nm in diameter are presented. Individual molecules are found to be able to cause multiple levels of conductance blockade upon traversing the pore. By analyzing these conductance blockades and their relative incidence as a function of nanopore diameter, it is concluded that the smallest conductance blockades likely correspond to molecules that translocate through the pore in predominantly head-to-tail fashion. The larger conductance blockades are likely caused by molecules that arrive at the nanopore entrance with many strands simultaneously. These measurements constitute the first demonstration that single-stranded RNA can be captured in solid-state nanopores that are smaller than the diameter of double-stranded RNA. These results further the understanding of the conductance blockades caused by nucleic acids in solid-state nanopores, relevant for future applications, such as the direct determination of RNA secondary structure.     

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.     

高放废物中^238Pu,^240Pu,^242Pu在聚变—裂变混合堆内的嬗变研究

从中子学角度研究了高放废物中２３８Ｐｕ、２４０Ｐｕ、２４２Ｐｕ在聚变一裂变混合堆内擅变的可行性。选取２３３Ｕ做中子增殖剂，对四个不同燃料组分的快谱包层进行了设计，利用输运一燃耗程序ＢＩＤＥＡＹ对所选方案进行了计算分析，结果表明：用２３３Ｕ做中子增殖剂，在聚变一裂变混合堆快谱包层内擅变２３８Ｐｕ、２４０Ｐｕ、２４２Ｐｕ是安全可行的。     

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.