犬2型腺病毒通用载体的构建及鉴定

为了获得能够携带较大外源基因的犬2型腺病毒E3区缺失性载体,以犬2型腺病毒全基因组质粒pPolyⅡ-CAV-2及E3区重组质粒pVAX-E3为基础,缺失1381bp的E3区片段(92.6%的E3区全序列),插入Linker-NF(内含NotⅠ、ClaⅠ、FseⅠ多克隆位点),获得重组载体质粒pPolyⅡ-CAV-2-ΔE3（NF）(31.9kb)。以AscⅠ和PmeⅠ双酶切,游离重组基因组,在脂质体Lipofectamine^TM 2000介导下,转染MDCK细胞系,获得了E3区缺失的重组病毒CAV-2-ΔE3（NF）。通过病毒的形态学观察,血凝性、生长特性、感染性实验证明,该重组病毒与母源病毒没有差异。重组病毒CAV-2-ΔE3（NF）可以作为载体表达外源基因,其外源基因插入片段不小于3.3kb。     

葛根素对糖尿病心肌细胞的保护及其机制研究

观察葛根素（Puerarin）对链脲佐菌素（streptozotocin,STZ）诱导的糖尿病大鼠心肌细胞的保护作用,并探讨血小板反应素1（Thrombospondin-1,TSP-1）的表达改变及其作用。雄性SD大鼠45只随机分为三组（n=15）：糖尿病组和葛根素治疗组采用一次腹腔注射链脲佐菌素（STZ）65mg／kg制备糖尿病模型,其中葛根素治疗组于造模后葛根素腹腔注射4周（100mg/kg/day）,正常对照组仅腹腔注射等量生理盐水（6ml／kg）,同样喂养4周。四周后各组大鼠处死。H—E染色及透射电子显微镜观察三组大鼠心肌细胞纤维显微结构和超微结构的病理改变．免疫组化和实时荧光定量PCR法观察大鼠心肌细胞中TSP-1蛋白和mRNA表达的变化．同时利用Langendorff离体心脏灌流法测定各组大鼠心室肌细胞功能。结果发现葛根素治疗组较糖尿病组大鼠的体重增加明显,同时血糖下降,有显著性差异（P〈0．01）。H—E染色显示糖尿病大鼠多处心肌肌丝紊乱伴少量炎症细胞浸润,电镜下发现有线粒体嵴消失溶解,肌丝排列紊乱等病理改变,而葛根素治疗组大鼠偶见上述病理变化。免疫组化显示葛根素治疗组心肌内TSP-1阳性细胞密度小于糖尿病大鼠,TSP-1 mRNA表达也比糖尿病大鼠要低。此外葛根素治疗组大鼠的左室收缩末压（LVSEP）、左心室舒张末期压（LVEDP）等心功能指标均明显低于正常组（P〈0．01）,但较糖尿病组有显著改善（P〈0．01）。上述结果显示葛根素能保护糖尿病大鼠心肌细胞的高糖损伤和维持心室肌细胞的功能,而该机制可能与抑制心肌细胞TSP-1表达的水平有关。     

Differential expression and regulation of expansin gene family members during fruit growth and development of ‘Shijia’ longan fruit

In this work, five expansin cDNAs (DlExp1–5) from ‘Shijia’ longan fruit were isolated and characterized. Moreover, the expression profiles of five expansin genes and the effect of naphthalene acetic acid (NAA) and thidiazuron (TDZ) on their expressions were investigated. The results showed that five expansins exhibited different expression patterns during fruit growth and development. DlExp1 was constitutively expressed in the pericarp while the levels of DlExp1 mRNA in the aril were very high at early stage of fruit development, and decreased gradually from 28 to 77 days after anthesis (DAA). DlExp2 and DlExp4 were related to the growth of pericarp, whereas the expression of DlExp2 and DlExp5 in the aril decreased from 28 to 77 DAA. In addition, NAA and TDZ applied at the stage of rapid pericarp (21 DAA) or aril growth (56 DAA) increased the accumulations of DlExp1 and DlExp2 mRNA in the pericarp and aril, while NAA and TDZ had no or little effect on the accumulations of DlExp3, DlExp4 and DlExp5. DlExp1 and DlExp2 also accumulated highly in rapidly growing tissues, such as young stems and leaves. These findings indicated that Exp genes played a different role in longan fruit growth and showed different response to plant growth substances.     

Enhancement of Astragalus polysaccharide on the immune responses in pigs inoculated with foot-and-mouth disease virus vaccine

The effects of Astragalus polysaccharides (APS) on the immune response in pigs immunized with foot-and-mouth disease virus (FMDV) vaccine were investigated. Fifteen pigs were randomly divided into five groups. Four groups were vaccinated with a FMDV inactivated vaccine. Pigs in three experimental groups were administered varying doses of APS (APS1, 5 mg/kg; APS2, 10 mg/kg; APS3, 20 mg/kg). The influence of APS on the number of CD3⁺CD4⁻CD8⁺ cytotoxic T cells, CD3⁺CD4⁺CD8⁺ T helper memory cells, and CD3⁻CD4⁻CD8⁺ natural killer cells among peripheral blood lymphocytes (PBL) in the three APS groups were significant compared to the vaccine group. In vitro stimulation of PBL by Con A and LPS in APS groups induced a stronger proliferative response at 2 and 6 weeks post-inoculation (PI). APS markedly increased the titer of FMDV-specific antibody in a dose-dependent manner, and up-regulated mRNA expression of IFN-γ and IL-6. APS could potentially be used as an immunomodulator for a FMDV vaccine and provide better protection against FMDV.     

Identification of diagnostic biomarkers for infection in premature neonates

Infection is a leading cause of neonatal morbidity and mortality worldwide. Premature neonates are particularly susceptible to infection because of physiologic immaturity, comorbidity, and extraneous medical interventions. Additionally premature infants are at higher risk of progression to sepsis or severe sepsis, adverse outcomes, and antimicrobial toxicity. Currently initial diagnosis is based upon clinical suspicion accompanied by nonspecific clinical signs and is confirmed upon positive microbiologic culture results several days after institution of empiric therapy. There exists a significant need for rapid, objective, in vitro tests for diagnosis of infection in neonates who are experiencing clinical instability. We used immunoassays multiplexed on microarrays to identify differentially expressed serum proteins in clinically infected and non-infected neonates. Immunoassay arrays were effective for measurement of more than 100 cytokines in small volumes of serum available from neonates. Our analyses revealed significant alterations in levels of eight serum proteins in infected neonates that are associated with inflammation, coagulation, and fibrinolysis. Specifically P- and E-selectins, interleukin 2 soluble receptor alpha, interleukin 18, neutrophil elastase, urokinase plasminogen activator and its cognate receptor, and C-reactive protein were observed at statistically significant increased levels. Multivariate classifiers based on combinations of serum analytes exhibited better diagnostic specificity and sensitivity than single analytes. Multiplexed immunoassays of serum cytokines may have clinical utility as an adjunct for rapid diagnosis of infection and differentiation of etiologic agent in neonates with clinical decompensation.     

杜鹃花科植物活性成分及作用机制研究进展

杜鹃花科(Ericaceae)植物约有54属,1700余种,分布极广,主产于南、北半球的温带和北半球的亚寒带。中国约有15属550余种,其中有毒品种9属100种以上,集中于金叶子属(Cribiodendron)、米饭花属(Lyonia)、马醉木属(Pieris)和杜鹃花属(Rhododendron)〔1〕。中国西南山区被认为是全国也是全世界的杜鹃花属分布中心,不少种被引种到欧美,成为当地著名庭园花卉的亲本〔2〕。杜鹃花科植物或具极高的欣赏价值,或有很高的药用价值,其广阔的开发应用前景已引起国内外的重视。笔者综述了近年来关于杜鹃花科植物活性成分、构效关系、生物活性及作用机制方…     

Expression and purification of the catalytic domain of human vascular endothelial growth factor receptor 2 for inhibitor screening

Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, can act in tumor-induced angiogenesis by binding to specific receptors on the surface of endothelial cells. One such receptor, VEGFR-2/KDR, plays a key role in VEGF-induced angiogenesis. Here, we expressed the catalytic domain of VEGFR-2 as a soluble active kinase using Bac-to-Bac expression system, and investigated correlations between VEGFR-2 activity and enzyme concentration, ATP concentration, substrate concentration and divalent cation type. We used these data to establish a convenient, effective and non-radioactive ELISA screening technique for the identification and evaluation of potential inhibitors for VEGFR-2 kinase. We screened 200 RTK target-based compounds and identified one (TKI-31) that potently inhibited VEGFR-2 kinase activity (IC50=0.596 microM). Treatment of NIH3T3/KDR cells with TKI-31 blocked VEGF-induced phosphorylation of KDR in a dose-dependent manner. Moreover, TKI-31 dose-dependently suppressed HUVEC tube formation. Thus, we herein report a novel, efficient method for identifying VEGFR-2 kinase inhibitors and introduce one, TKI-31, that may prove to be a useful new angiogenesis inhibitor.     

南瓜属4个栽培种种子蛋白质电泳分析

通过聚丙烯酰胺凝胶电泳(PAGE)对南瓜属4个栽培种中国南瓜、印度南瓜、美洲南瓜及黑籽南瓜的12个品种种子蛋白组分进行了分析。结果显示,黑籽南瓜种子以盐溶蛋白条带居多;其它3个栽培种以水溶蛋白条带居多,其次为盐溶蛋白;4个栽培种酸溶蛋白条带均较少;醇溶蛋白未分离得到蛋白谱带。水溶、盐溶、酸溶蛋白电泳图谱显示各栽培种间蛋白谱带差异明显,同一栽培种品种间蛋白谱带较为一致。