脱氧核酶的应用研究进展

核酸新功能的发现进一步加深了人们对DNA的认识.脱氧核酶具有RNA切割作用、DNA切割作用、金属螯合作用、过氧化物酶活性、DNA激酶活性以及DNA连接酶活性等多种催化功能.通过对功能核酸的研究,开辟了脱氧核酶在实际应用方面的新方向.     

脱氧核酶电化学生物传感器高灵敏测定铅离子

基于铅离子脱氧核酶对铅离子的特异性识别作用,研制了用于测定铅离子的高灵敏电化学生物传感器。该传感器以固定于电极表面、末端标记了电信号基团的脱氧核酶作为识别元素和信号探针。当传感界面与含有铅离子的溶液接触时,脱氧核酶的酶链在铅离子的辅助作用下将修饰了电信号基团的底物链切断并使其脱离电极表面,从而使电极表面电信号分子的电流减小,产生用于铅离子定量检测的电信号。在0.5~12.5μg/L的浓度范围内,检测信号与Pb~(2+)浓度呈良好的线性关系,Pb~(2+)的检出限为0.3μg/L。同时,该电化学生物传感器对Pb~(2+)的检测表现出良好的特异性和选择性。     

10-23脱氧核酶的研究进展

10-23脱氧核酶(Deoxyribozyme,DRz)是利用体外分子进化技术筛选得到的一种具有催化功能的单链DNA片段,具有高效的催化活性及特异的序列识别能力,能催化RNA特定部位的切割反应,抑制相应蛋白的表达.本文就10-23 DRz的结构、活性影响因素、反应机制、生物特性及应用的研究进展作一综述.     

非标记脱氧核酶构象改变荧光法检测铀酰离子

在p H 5.5 MES缓冲溶液中,当体系中不存在铀酰离子时,SYBR GreenⅠ(SG)能通过嵌入和小沟结合方式与脱氧核酶作用,导致荧光增强;当铀酰离子存在时,脱氧核酶的r A碱基处断裂,游离出单链,此时SG与单链DNA作用减弱,荧光信号降低,且体系的荧光强度变化值与铀酰离子浓度在1.73×10-9~4.40×10-8mol/L范围内呈良好线性关系,线性回归方程为ΔF=108.99C(×10-8mol/L)+79.22,相关系数r=0.990。根据空白管的标准偏差Sb和标准曲线的斜率k算出LOD为5.2×10-10mol/L。该方法简便、选择性好、灵敏度高。     

非标记脱氧核酶构象改变荧光法检测铀酰离子

在p H 5.5 MES缓冲溶液中,当体系中不存在铀酰离子时,SYBR GreenⅠ(SG)能通过嵌入和小沟结合方式与脱氧核酶作用,导致荧光增强;当铀酰离子存在时,脱氧核酶的r A碱基处断裂,游离出单链,此时SG与单链DNA作用减弱,荧光信号降低,且体系的荧光强度变化值与铀酰离子浓度在1.73×10-9⁴.40×10-8mol/L范围内呈良好线性关系,线性回归方程为ΔF=108.99C(×10-8mol/L)+79.22,相关系数r=0.990。根据空白管的标准偏差Sb和标准曲线的斜率k算出LOD为5.2×10-10mol/L。该方法简便、选择性好、灵敏度高。     

基因活化剂抑制龙眼冲梢和提高产量品质的效果

在龙眼牙芽生理,形态化期(１,２月份)各喷旋基因活化剂２５０,５００倍液１,２次,前者增产３７８.５kg/667m^2和７９１kg/667m^2,效果十分显著,后者增产５５.1kg/667m^2, 和１０７.９kg/667m^2,效果比较显著,生物学上表现在“冲梢“量和春梢营养枝抽发量少,着果好,果穗长度缩短,分枝数增多,单穗结果增加１２３-３６个,单穗果重增重７４４.２-２１４.３g,小果数减少３１.２５%-１.４６%,果实品质与对照比可食率增加０.６７%-４.４８%,可溶性固形物提高８.０８-１.９８个百分点.     

基于脱氧核酶的生物传感器的实际应用

自近30年前发现脱氧核酶以来,脱氧核酶已广泛应用于生物传感领域。电化学法、荧光、比色法、电化学发光、化学发光、光电化学等检测方法在基于脱氧核酶的生物传感器中广泛应用于生物样品方面的检测。主要讨论了现有的基于脱氧核酶的生物传感器的几种常见检测方法在检测生物样品方面的实际应用,并且讨论了几种检测方法的优缺点,并指出了现有研究的不足之处。     

酸雾抑制方法的研究与进展

由于酸雾对生产工人的健康、大气环境和生产设备等均有严重损害,如何有效抑制酸雾一直是困扰着冶金、金属制品加工等行业的难题。作者按物理法和化学法分类介绍了目前国内外酸雾抑制方法的现状以及研究进展,并着重分析了小球覆盖法及泡沫覆盖法的使用现状。通过综合各个抑雾方法,阐明了现有的抑制酸雾技术的不足,并对一种新型低密度、无渗透性的高分子微球在酸雾抑制领域的应用前景进行了展望。     

天然产物对酪氨酸酶的抑制及抑制机理的研究进展

介绍了酪氨酸酶的结构及酶促褐变机制；重点阐述了对酪氨酸酶抑制的特征及抑制机理。酪氨酸酶的抑制特征为可逆抑制，分为竞争性、非竞争性、混合型及缓慢结合型4种。其抑制的主要机理分别为：底物类似物结合酶的活性中心从而抑制酶活性；抑制剂与酶活性中心以外的氨基酸残基结合及抑制剂对过氧自由基的清除作用；抑制剂对酶活性中心的内源桥基的影响；抑制剂与酶快速形成复合物，此后经历一个缓慢的可逆异构化过程。     

水合物动力学抑制研究现状

着重评述了二类低用量水合物抑制剂,即动力学抑制剂和防聚剂的抑制机理方面的理论研究成果,以及抑制性能方面的实验研究成果。简要介绍了低用量水合物抑制剂的应用状况。根据水合物动力学抑制的研究现状,指出了可作为今后研究重点的5个方面。     

Thrombin-Binding Aptamer with Inversion of Polarity Sites (IPS): Effect on DNAzyme Activity and Anticoagulant Properties

In this work we examined the properties of thrombin-binding aptamer (TBA) modified by the introduction of inversion of polarity sites (IPS) in order to assess the effect of modification on the activation of TBA to serve as DNAzyme with peroxidase-like activity. Two oligonucleotides were designed to possess one (IPS1) or three (IPS2) inversion sites. TBA typically forms antiparallel G-quadruplexes with two G-tetrads, which exhibits very low DNAzyme peroxidise activity. DNAzyme activity is generally attributed to parallel G-quadruplexes. Hence, inversion of polarity was introduced in the TBA molecule to force the change of G-quadruplex topology. All oligonucleotides were characterized using circular dichroism and UV-Vis melting profiles. Next, the activity of the DNAzymes formed by studied oligonucleotides and hemin was investigated. The enhancement of peroxidase activity was observed when inversion of polarity was introduced. DNAzyme based on IPS2 showed the highest peroxidase activity in the presence of K⁺ or NH₄⁺ ions. This proves that inversion of polarity can be used to convert a low-activity DNAzyme into a DNAzyme with high activity. Since TBA is known for its anticoagulant properties, the relevant experiments with IPS1 and IPS2 oligonucleotides were performed. Both IPS1 and IPS2 retain some anticoagulant activity in comparison to TBA in the reaction with fibrinogen. Additionally, the introduction of inversion of polarity makes these oligonucleotides more resistant to nucleases.     

Controlled Release of a Dual Zinc-Sensing and Gene Regulating Small Molecule from DNA Micelles

Intracellular zinc ions are essential for various biological cell processes and are often dysregulated in many diseases de-pending on their location, protein binding affinity, and concentration in the cell. Due to their prevalence in diseases, it is important to not only effectively sense but chelate the often excess amount of zinc in a cell to alleviate further disease progression. N, N, N′, N′-tetrakis (2-pyridinylmethyl)-1,2-ethanediamine (TPEN) is a selective zinc chelator but its water-insoluble nature and general cytotoxicity limit its therapeutic potential. To address these challenges, TPEN loaded nucleic acid nanocapsules (TL-NANs) were synthesized, and its dual ability to sense and suppress zinc levels intracellularly were evaluated. Additionally, TL-NANs were incubated in lung cells and shown to down regulate Eotaxin, a protein up-regulated during asthma, at significantly reduced concentrations of TPEN showcasing the therapeutic potential of this drug for asthma.     

Regulation of Gene Expression by Telomere Position Effect

Many diseases that involve malignant tumors in the elderly affect the quality of human life; therefore, the relationship between aging and pathogenesis in geriatric diseases must be under-stood to develop appropriate treatments for these diseases. Recent reports have shown that epigenetic regulation caused by changes in the local chromatin structure plays an essential role in aging. This review provides an overview of the roles of telomere shortening on genomic structural changes during an age-dependent shift in gene expression. Telomere shortening is one of the most prominent events that is involved in cellular aging and it affects global gene expression through genome rearrangement. This review provides novel insights into the roles of telomere shortening in disease-affected cells during pathogenesis and suggests novel therapeutic approaches.     

RNA干扰P115基因对胃癌细胞巨噬细胞移动抑制因子表达的抑制作用

目的构建高尔基体转运蛋白P115基因shRNA表达载体,探讨P115基因沉默对胃癌细胞株BGC-823中巨噬细胞移动抑制因子(Macrophage migration inhibitory factor,MIF)表达的影响。方法设计4对针对P115基因的shRNA序列,构建重组表达质粒,转染高表达P115的胃癌细胞株BGC-823。RT-PCR、Western blot和免疫细胞化学检测P115及MIF的mRNA和蛋白的表达。结果 4个P115基因shRNA质粒经单酶切和测序证实构建正确;转染BGC-823细胞后,均能抑制P115基因的表达,其中以pGPU6/GFP/Neo-shP115-2的沉默效果最好,其对P115基因mRNA表达的抑制率为75.07%,对P115蛋白表达的抑制率为70.97%;转染pGPU6/GFP/Neo-shP115-2后,MIF基因的mRNA及蛋白表达水平均明显降低(P<0.05)。结论 P115基因沉默后,BGC-823细胞MIF的表达明显降低,提示P115可能参与调节胃癌细胞MIF的表达,P115基因可作为研究胃癌发生发展分子机理的新靶点。     

RNA干扰技术对MCF-7细胞Aurora-A基因表达的影响

目的构建针对Aurora-A基因的特异性小干扰RNA(siRNA)真核表达载体,并探讨其对人乳腺癌细胞株MCF-7中Aurora-A基因表达的抑制作用。方法将具有短发夹结构的2条DNA序列,经退火形成互补双链,再克隆至载体pGCsi-U6/Neo/GFP中,转化大肠杆菌DH5α,提取质粒进行序列测定。并通过脂质体法转染至MCF-7细胞中,48h后观察转染效率,并采用RT-PCR及Westernblot法检测siRNA对MCF-7细胞Aurora-A基因mRNA及蛋白表达的影响。结果测序鉴定证实目的寡核苷酸片段已被克隆至pGCsi-U6/Neo/GFP载体中,Aurora-A-siRNA质粒转染MCF-7细胞48h后转染效率约为60%,与对照组比较,Aurora-A-siRNA质粒转染的细胞Aurora-A基因mRNA和蛋白的表达均明显下降。结论已成功构建了Aurora-A-siRNA真核表达质粒,其能明显抑制MCF-7细胞Aurora-A基因的表达,为进一步研究Aurora-A基因的功能奠定了基础,并可能为肿瘤的生物学治疗提供新的方法。     

RNA干扰BC047440基因表达对HCT-8细胞增殖能力的影响

目的探讨RNA干扰结肠癌相关基因BC047440的表达对结肠癌细胞HCT-8增殖能力的影响。方法将BC047440基因shRNA干扰质粒pGPU6/GFP/Neo-BC047440-331(a1)、pGPU6/GFP/Neo-BC047440-451(a2)、pGPU6/GFP/Neo-BC047440-615(a3)、pGPU6/GFP/Neo-BC047440-756(a4)采用脂质体法转染结肠癌细胞HCT-8,荧光显微镜观察转染效率,荧光定量PCR法检测细胞BC047440基因mRNA的表达水平,MTT法分析细胞的增殖活性。结果细胞的转染效率为53%;干扰质粒a1、a2、a3、a4转染的HCT-8细胞BC047440基因mRNA的表达水平分别下降了36.1%、47.0%、53.8%和63.3%,a4质粒的干扰效率最高;细胞的增殖能力也明显下降。结论 BC047440基因RNA干扰质粒可明显抑制HCT-8细胞BC047440基因mRNA的表达及细胞的增殖,可能成为抑制结肠癌细胞生长的新基因。     

表达鸡新城疫病毒血凝素-神经氨酸酶基因的重组腺病毒对肝癌细胞HepG-2的抑制作用

目的探讨表达鸡新城疫病毒(NDV)血凝素-神经氨酸酶(HN)基因的重组腺病毒Ad-HN对人肝癌细胞HepG-2的抑制作用。方法应用Ad-HN感染HepG-2细胞,采用MTT法检测Ad-HN对HepG-2细胞增殖的抑制作用;AnnexinV染色法结合流式细胞仪检测细胞凋亡;AO/EB染色法和DAPI染色法对Ad-HN感染的肿瘤细胞及细胞核进行形态学观察;底物显色法检测Caspase1、Caspase3、Caspase6和Caspase8活性的变化。结果Ad-HN能够有效抑制HepG-2细胞的增殖,当感染剂量为100MOI,作用时间为48h时,Ad-HN对HepG-2细胞增殖的抑制率达峰值(17.20%～35.04%);AnnexinV染色结果显示,Ad-HN感染48h后HepG-2细胞凋亡率为29.39%;Ad-HN感染导致HepG-2细胞呈现细胞浓缩、细胞核皱缩进而碎裂等凋亡特征;Ad-HN感染可显著上调HepG-2细胞的Caspase酶活性。结论Ad-HN能够诱导HepG-2细胞凋亡,并对HepG-2细胞产生抑制效应。     

人MCIR基因在杆状病毒-昆虫细胞表达系统中的表达

目的利用杆状病毒表达系统进行人恶性黑色素瘤A375细胞MCIR基因在Sf9昆虫细胞中的表达。方法以pMD18-T-MCIR为模板，利用PCR方法扩增人MCIR基因，将MCIR基因连接到pfastBacl质粒上，与穿梭载体DH10Bac转座，获得Bacmid-MCIR质粒后，通过脂质体介导，转染Sf9细胞，使其表达重组杆状病毒，经SDS-PAGE检测表达产物，放射受体分析法检测目的蛋白MCIR活性。结果Bacmid-MCIR质粒转染Sf9细胞后的SDS-PAGE图谱，在相对分子质量为35000处有一条新生蛋白带。放射受体分析结果显示表达的蛋白与标记配体特异亲和，具有生物学活性。结论MCIR基因成功在真核细胞中得到表达。     

Effects of Titanium Dioxide Nanoparticle Aggregate Size on Gene Expression

Titanium dioxide (titania) nanoparticle aggregation is an important factor in understanding cytotoxicity. However, the effect of the aggregate size of nanoparticles on cells is unclear. We prepared two sizes of titania aggregate particles and investigated their biological activity by analyzing biomarker expression based on mRNA expression analysis. The aggregate particle sizes of small and large aggregated titania were 166 nm (PDI = 0.291) and 596 nm (PDI = 0.417), respectively. These two size groups were separated by centrifugation from the same initial nanoparticle sample. We analyzed the gene expression of biomarkers focused on stress, inflammation, and cytotoxicity. Large titania aggregates show a larger effect on cell viability and gene expression when compared with the small aggregates. This suggests that particle aggregate size is related to cellular effects.     

毛尖茶叶中的黄酮类化合物对脂氧合酶的抑制性

采用紫外可见光分光光度法,以亚油酸钠为底物,研究了都匀毛尖茶叶中的黄酮类化合物对大豆脂氧合酶活性的抑制作用。结果表明,毛尖茶叶中的黄酮类化合物的提取率为5.227%,毛尖茶叶中的黄酮类化合物对大豆脂氧合酶有一定的抑制效果,并且随着黄酮类化合物的浓度的增加抑制效果增强。