稳定表达烟酸受体GPR109A的中国仓鼠卵巢细胞株的建立

目的 将GPR109A基因插入pEGFP-N3载体中构建重组质粒pEGFP-GPR109A,并建立稳定表达烟酸受体GPR109A的中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞株.方法 构建重组质粒pEGFP-GPR109A,重组质粒转化大肠杆茵DH5a,重组质粒经PCR、酶切、测序验证正确后,应用脂质体转染技术将该质粒导入CHO细胞,用抗生素G418筛选稳定表达的细胞.倒置荧光显微镜观察克隆细胞株荧光信号,RT-PCR检测GPR109A基因mRNA表达,Westem Blotting检测绿色荧光蛋白与烟酸受体GPR109A的融合蛋白(GFP-GPR109A)的表达,激光共聚焦显微镜观察融合蛋白的细胞定位.结果 pEGFP-GPR109A真核表达质粒构建正确,融合蛋白GFP-GPR109A在CHO细胞中稳定表达,表达的融合蛋白主要定位于细胞膜.结论 成功构建pEGFP-GPR109A真核表达载体并建立其稳定表达的CHO细胞株;该细胞株的建立有助于GPR109A的更多生理病理功能研究,也为下一步抗动脉粥样硬化的药物筛选奠定了基础.     

Evaluation of Multicomponent Non-viral Vectors for Liver Directed Gene Delivery

Multicomponent, non-viral gene delivery vehicles are designed to have as a minimum, a DNA binding component, and a cell recognition component for specific delivery to target cells. The DNA binding component cannot only bind, but also protect DNA from serum degradation, and tends to condense DNA to sizes that can be taken up by receptor-mediated processes of target cells. Generally, cationic peptides, single chained, e.g. poly- l -lysine or branched polymers or synthetic peptides with DNA binding properties are used for DNA binding components. Ligands for binding to receptors on cell surfaces can be covalently linked to the DNA binding component. Multicomponent, non-viral vectors have been successfully used to deliver genes into cells in vitro and in vivo. Improvements have been made to the non-viral carriers resulting in increased solubility of DNA/carrier complexes and longer survival in serum. Improvements have also been made by incorporating fusogenic/lysosomolytic components that enable DNA/carrier complexes to escape intracellular degradation and enhance the levels and duration of expression of genes in vitro and in vivo.     

Growth of tick-borne encephalitis virus (European subtype) in cell lines from vector and non-vector ticks

We undertook a comparative study of the susceptibility of different tick cell lines to infection with the European subtype of tick-borne encephalitis virus (TBEV), prototype strain Neudoerfl. The growth of TBEV was investigated in lines derived from vector Ixodes ricinus L. ticks (IRE/CTVM18, 19, and 20), as well as non-vector ticks, namely Ixodes scapularis Say (IDE2), Boophilus microplus Canestrini (BME/CTVM2), Hyalomma anatolicum anatolicum Koch (HAE/CTVM9), Rhipicephalus appendiculatus Neumann (RA-257) and recently established and herein described lines from the argasid tick Ornithodoros moubata Murray (OME/CTVM21 and 22). All the tick cell lines tested were susceptible to infection by TBEV and the virus caused productive infection without any cytopathic effect. However, there was a clear difference between the TBEV growth in vector and non-vector cell lines, since I. ricinus cell lines produced 100-1000-fold higher virus yield than the non-vector cell lines. The lowest virus production was observed in O. moubata and R. appendiculatus cell lines.     

PHLEBOTOMINE FAUNA (DIPTERA: PSYCHODIDAE) IN AN AREA OF FISHING TOURISM IN CENTRAL-WESTERN BRAZIL

The aim of this study was to identify behavioral aspects of the sandfly fauna of a fishing tourism area in the municipality of Bonito (MS). Monthly captures were undertaken from December 2009 to November 2010, using automatic CDC type light traps, from 18h00 to 06h00, in a forested area, a savannah area, peridomiciles and animal shelters near peridomiciliary areas. Nyssomyia whitmani was the most frequent out of a total of 6,699 specimens collected, belonging to 16 species, followed by Psathyromyia bigeniculata and Lutzomyia longipalpis, found in all the environments investigated, though in their greatest numbers in the animal shelters. Ny. whitmani exhibited its highest frequencies during the dry months, coincident with the fishing season, when the risk of transmission of cutaneous leishmaniasis for tourists and inhabitants increases. Noteworthy was the finding of two species naturally infected by flagellates: Ny. whitmani and Pa. bigeniculata. The local population and visiting tourists should be warned of the threat posed by leishmaniasis and the health authorities alerted to the need for adopting environmental sanitary measures, especially regarding such animal shelters as they seem to provide favorable conditions to the proliferation, maintenance and breeding opportunities of phlebotomines.     

EGFL7原核表达载体的构建及鉴定

【目的】构建类表皮生长因子域7（EGFL7）原核表达载体,并诱导其表达、纯化及鉴定目的蛋白。【方法】以人肝癌细胞系 HepG2总RNA为模板,PCR扩增 EGFL7基因,克隆至原核表达载体pET‐21a（＋）中,转化大肠杆菌 BL21（DE3）,IPTG 诱导表达。 His‐tag 磁珠纯化重组蛋白 EGFL7,SDS‐PAGE 鉴定。【结果】克隆目的基因序列正确,未发生碱基突变;重组表达质粒pET21a（＋）‐TRX‐EGFL7经双酶切鉴定构建正确;表达的重组蛋白相对分子量为80kD ,与预期相符。【结论】成功在大肠杆菌BL21（DE3）中表达并纯化了EGFL7重组蛋白,为后续研究奠定了基础。     

Gene therapy strategies for the treatment of cancer. A review

This review summarizes current trends in cancer gene therapy. The diversity of approaches and rapid application of technical improvements reflects the interest in this emerging field. Major topics include the restoration of tumor suppressor genes and inhibition of oncogenes, stimulation of the immune response against tumors, and sensitization of cancer cells to systemically administered prodrugs. Special attention is focused on methodstumor targeting and the development of new vectors.