进口散装废金属原料及其船舶携带媒介生物调查研究

目的通过对进口散装废金属原料及其船舶媒介生物的调查,为开展口岸媒介生物防治提供科学依据。方法按照《国境口岸及出入境交通工具医学媒介生物监测规定》方法,选取210批次散装废金属原料,以及767艘次装运废金属原料船舶进行医学媒介生物调查,并对调查数据进行统计分析。结果在进口散装废金属原料中未截获医学媒介生物,运载船舶发现医学媒介107艘次,阳性率13.95%;在103艘船舶(13.42%)上发现2种蝇类,共1 174只;在4艘船舶上发现蟑螂,共265只,阳性率0.52%;鼠类、蚊类、蚤类未发现。结论使用目前常规媒介监测手段难以发现进口散装废金属原料携带医学媒介情况。装载散装废金属原料船舶有较高的医学媒介生物携带率,需要加强对该类船舶的卫生检疫工作。     

Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs

Since the development of infectious cDNA clones of viral RNA genomes and the means of delivery of the in vitro-synthesized RNA into cells, alphaviruses have become an attractive system for expression of heterologous genetic information. Alphaviruses replicate exclusively in the cytoplasm, and their genetic material cannot recombine with cellular DNA. Alphavirus genome-based, self-replicating RNAs (replicons) are widely used vectors for expression of heterologous proteins. Their current design relies on replacement of structural genes, encoded by subgenomic RNAs (SG RNA), with heterologous sequences of interest. The SG RNA is transcribed from a promoter located in the alphavirus-specific RNA replication intermediate and is not further amplified. In this study, we have applied the accumulated knowledge of the mechanism of alphavirus replication and promoter structures, in particular, to increase the expression level of heterologous proteins from Venezuelan equine encephalitis virus (VEEV)-based replicons. During VEEV infection, replication enzymes are produced in excess to RNA replication intermediates, and a large fraction of them are not involved in RNA synthesis. The newly designed constructs encode SG RNAs, which are not only transcribed from the SG promoter, but are additionally amplified by the previously underused VEEV replication enzymes. These replicons produce SG RNAs and encoded proteins of interest 10- to 50-fold more efficiently than those using a traditional design. A modified replicon encoding West Nile virus (WNV) premembrane and envelope proteins efficiently produced subviral particles and, after a single immunization, elicited high titers of neutralizing antibodies, which protected mice from lethal challenge with WNV.Alphaviruses are a group of enveloped viruses with a positive-strand RNA genome that replicate in most commonly used cell lines to titers exceeding 10¹⁰ infectious units (inf.u)/mL (1, 2). Upon infection, the genomic RNA serves as a template for translation of viral nonstructural proteins that form replication complexes (3). Within a few hours postinfection, these complexes synthesize large amounts of viral genomic and subgenomic (SG) RNA (3). The SG RNA is transcribed from the SG promoter and serves as a template for translation of viral structural proteins: capsid, E2 and E1, which ultimately assemble with genomic RNA into infectious viral particles. This highly efficient virus-specific RNA and protein synthesis, coupled with the availability of infectious cDNA clones, have made alphaviruses an attractive system for designing self-replicating vectors for delivery and expression of heterologous genetic information. The most widely used alphavirus-based expression systems are based on replacement of viral structural genes by a gene(s) of interest (4). These modified viral genomes, termed replicons, can be synthesized in vitro and delivered into cells either by transfection or in infectious viral particles, which deliver essentially every packaged RNA molecule into the cells both in vivo and in vitro.In recent years, significant progress has been made in our understanding of the mechanism of alphavirus replication. Detailed studies have elucidated the structure and function of the RNA promoters, critical aspects of virus–host cell interactions, and the composition of the replication complexes (5–12). These mechanistic studies of alphavirus replication raised the question of whether we are using their entire expression potential, and whether the traditional replicon design can be further improved to achieve higher levels of heterologous protein production. In this project, we sought to apply the latest advances in understanding of alphavirus RNA replication to design a new generation of Venezuelan equine encephalitis virus (VEEV) genome-based expression systems. The distinguishing feature of these constructs is the modification of the SG RNAs. These SG RNAs have been engineered to contain the cis-acting promoter elements, which are normally present at the 5′ end of the viral genome and mediate genomic RNA replication (8, 13, 14). Thus, in these newly designed VEEV replicons (VEErep), the SG RNAs were not only transcribed from the SG promoter, but were capable of replication/amplification by the VEEV replication complexes. As a result, the heterologous gene expression was more efficient than that of the existing constructs, which use replicons with the standard SG RNAs. The expression level of heterologous protein encoded by the improved replicons was also found to be dependent on coexpression of VEEV capsid protein. The VEEV replicons, which use both amplification of the SG RNA and express capsid protein, provide a platform for development of a variety of more efficient expression systems and have numerous applications. To illustrate this, we have generated a VEEV replicon encoding the premembrane and envelope (prM/E) proteins of West Nile virus (WNV). Particles containing the newly designed replicons induced high levels of WNV E protein expression in vitro and elicited robust protective immunity in mice.     

The effect of serum in culture on RNAi efficacy through modulation of polyplexes size

Serum in the culture medium is one crucial factor that compromises RNAi efficiency of non-viral vectors. However, mechanistic roles of serum in siRNA delivery remain unknown. In this work, we took one cationic polymer, pullulan chemically modified by spermine (termed as pullulan-spermine, Ps), as a siRNA carrier model to investigate the effects of serum on key steps in siRNA delivery including formation of Ps and siRNA polyplexes (Ps-siRNA), cellular uptake, lysosomal escape, and cytotoxicity. We demonstrate that low serum concentration (1.25% and 2.5%) in culture medium results in large particles of Ps-siRNA, while high serum concentration (10%–40%) leads to small particles of Ps-siRNA. The larger particles initiated the internalization of siRNA more effectively in comparison to the smaller ones. The engulfed Ps-siRNA particles mainly locate in lysosomes. The large particles exhibited stronger abilities of destabilizing lysosomes than that of the small particles as large Ps-siRNA particles contain more amines and subsequently elicit a stronger proton sponge effect which results in more effective lysosomal escape of siRNA. Despite the lower RNAi efficiency, the small particle of Ps-siRNA in the high serum medium generates much lower cytotoxicity. These findings explain why serum significantly affects RNAi and also propose a strategy for improving RNAi efficiency and safety by modulating serum concentration and enhancing lysosomal destabilization.     

PRS载体介导的表达CTGF-SiRNA逆转录病毒载体的构建

【目的】梅建表达人结缔组织生长因子（CTGF）小分子干扰RNA（small interfering RNA,SiR—NA）的PRS-CTGF-SiRNA逆转录病毒载体。【方法】根据SiRNA靶序列设计要求及PRS逆转录病毒载体特点分别设计含64bpDNA序列的4对寡核苷酸,并在体外合成。PRS载体用BgⅡ、HindⅢ双酶切完全后,分别与退火的4对寡核苷酸进行连接,连接后去载体自连,构建成表达人CTGF小分子干扰RNA的PRS-CTGF-SiRNA逆转录病毒载体。【结果】经酶切、连接后构建成的质粒称之为PRS-CTGF—SiRNA1～4,经酶切与测序证实构建成功,无任何碱基突变。【结论】成功构建了能表达人CTGF-SiRNA的PRS-CTGF—SiRNA逆转录病毒载体,为腹膜透析腹膜纤维化防治的体内、外研究提供一种可能有效的方法。     

HBV preS2真核载体的构建及其表达

【目的】扩增乙型肝炎病毒（HBV）前S2基因（preS2）,并构建其真核表达载体,为研究HBV树突状细胞疫苗奠定实验基础。【方法】从慢性乙型肝炎患者血清中提取HBVDNA,用聚合酶链反应技术扩增全长preS2基因,将其克隆到真核载体pcDNA3．1（＋）上,经PCR、酶切和DNA测序鉴定筛选阳性克隆,构建真核表达载体pcDNA3．1（＋）preS2,并将其转染巨噬细胞48h后,ELISA检测细胞裂解液中preS2的表达。【结果】从慢性乙型肝炎患者血清抽提的DNA中扩增得到约860bp大小的目的片段,preS2基因片段正确插入pcDNA3．1（＋）载体,得到HBVpreS2真核表达载体pcDNA3．1（＋）-pre2。【结论】成功地构建了舍preS2基因的真核表达载体pcDNA3．1（＋）-preS2,并能在巨噬细胞中表达目的蛋白HBVpreS2。     

第三代腺病毒载体的改良与应用

腺病毒载体是一种非常有效的转基因载体,具有高效率、低致病性、高滴度及不整合入宿主细胞染色体等优点,广泛应用于基因治疗.第一、二代腺病毒载体仍有许多不足之处,在其基础上发展了第三代腺病毒载体.第三代腺病毒载体与前者相比,具有低免疫原性及高容量等优点,在基因治疗中表现出独特优势.然而,由于辅助病毒污染的存在及病毒产量有待提高,第三代腺病毒载体的临床应用仍面临一定困难.     

Hydrophilic Random Cationic Copolymers as Polyplex-Formation Vectors for DNA

Research on the improvement and fabrication of polymeric systems as non-viral gene delivery carriers is required for their implementation in gene therapy. Random copolymers have not been extensively utilized for these purposes. In this regard, double hydrophilic poly[(2-(dimethylamino) ethyl methacrylate)-co-(oligo(ethylene glycol) methyl ether methacrylate] [P(DMAEMA-co-OEGMA)] random copolymers were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The copolymers were further modified by quaternization of DMAEMA tertiary amine, producing the cationic P(QDMAEMA-co-OEGMA) derivatives. Fluorescence and ultraviolet–visible (UV–vis) spectroscopy revealed the efficient interaction of copolymers aggregates with linear DNAs of different lengths, forming polyplexes, with the quaternized copolymer aggregates exhibiting stronger binding affinity. Light scattering techniques evidenced the formation of polyplexes whose size, molar mass, and surface charge strongly depend on the N/P ratio (nitrogen (N) of the amine group of DMAEMA/QDMAEMA over phosphate (P) groups of DNA), DNA length, and length of the OEGMA chain. Polyplexes presented colloidal stability under physiological ionic strength as shown by dynamic light scattering. In vitro cytotoxicity of the empty nanocarriers was evaluated on HEK293 as a control cell line. P(DMAEMA-co-OEGMA) copolymer aggregates were further assessed for their biocompatibility on 4T1, MDA-MB-231, MCF-7, and T47D breast cancer cell lines presenting high cell viability rates.     

关注致动脉粥样硬化的非传统危险因素

动脉硬化是指动脉管壁的增厚、变硬和弹性减低。动脉粥样硬化是动脉硬化的特定类型,指动脉管壁由于钙和脂肪物质在管壁堆积而变厚。尽管对动脉粥样硬化的发病机制至今尚未完全明了,但有较明确的原因即动脉粥样硬化的危险因素,包括传统和非传统危险因素,我们对传统危险因素已逐步认识。随着对血管危险因素的再认识,非传统危险因素开始被关注,这将对血管早期风险评价提供依据。现就非传统危险因素对动脉粥样硬化性血管病变的影响进行阐述。     

慢病毒介导的中性内肽酶对Aβ诱导的SK-N-SH细胞凋亡的影响

目的： 探讨脑中性内肽酶（NEP）外源表达对神经毒物质β淀粉样肽（Aβ）诱导SK-N-SH细胞凋亡的影响。方法: 采用脂质体法将含中性内肽酶NEP的四质粒系统慢病毒载体转染293FT包装细胞，制备高滴度病毒载体，感染Aβ处理的SK-N-SH细胞，用Western blotting方法检测NEP对Aβ的降解作用、MTT法检测细胞存活率、流式细胞术分析细胞凋亡情况、RT-PCR技术测定凋亡相关基因bcl-2及bax的表达及caspase-3活性检测。结果: 胞内高表达的NEP能够显著降解Aβ，其降解程度与NEP的表达量有剂量依赖关系。细胞存活率及流式细胞术检测结果显示NEP高表达可减轻Aβ诱导的细胞凋亡，细胞存活率于感染病毒后48 h最强，为对照的170%（P＜0.01）,NEP活性组的凋亡率为3.86%，低于对照组的6.41%；RT-PCR结果显示NEP对Aβ诱导的细胞凋亡保护作用可能是通过减少bax的表达，抑制了caspase-3的激活程度来实现的。结论: NEP可以降解Aβ，可以保护由Aβ沉积所致的神经细胞凋亡。