小鼠Fancd2os基因真核表达质粒的构建及其对人胚肾上皮细胞增殖和凋亡的影响

目的构建小鼠Fancd2os基因的真核表达质粒,并分析Fancd2os基因对人胚肾上皮细胞(HEK293T)增殖和凋亡的影响。方法以小鼠睾丸组织DNA为模板扩增Fancd2os基因c DNA全长片段,并克隆至真核表达载体p3×FLAG-CMV-14,构建重组真核表达质粒p3×FLAG-CMV-14-Fancd2os。经Lipofectamine TM 2000将重组真核表达质粒瞬时转染HEK293T细胞,分别采用RT-PCR及Western blot法检测转染细胞中Fancd2os基因m RNA转录及蛋白表达情况;经CCK8法和流式细胞术分别检测过表达Fancd2os对HEK293T细胞增殖能力及紫外线诱导HEK293T细胞凋亡的影响。结果经双酶切及测序鉴定,重组真核表达质粒p3×FLAG-CMV-14-Fancd2os构建正确;转染p3×Flag-CMV-14-Fancd2os的HEK293T细胞可见Fancd2os基因m RNA转录及其蛋白表达;过表达Fancd2os的HEK293T细胞的增殖活性显著下降(P0.05);过表达Fancd2os可显著促进紫外线诱导的HEK293T细胞凋亡(P0.01)。结论成功构建了重组真核表达质粒p3×Flag-CMV-14-Fancd2os,过表达Fancd2os可抑制HEK293T细胞的增殖,促进其凋亡。     

JTV1基因真核表达质粒的构建及其在K562细胞中的表达

目的 构建JTV1基因真核表达质粒并稳定转染人白血病细胞系K562,检测转染细胞中JTV1基因mRNA和蛋白的表达水平及其对K562细胞增殖的影响。方法从人外周血单个核细胞中克隆JTV1基因,并将其插入pcDNA3.1表达载体中,构建真核重组表达质粒pcDNA3.1-JTV1,经脂质体介导转染K562细胞,采用RT-PCR和Western blot法鉴定转染细胞中JTV1基因mRNA和蛋白的表达水平;MTT法检测JTV1稳定表达对K562细胞增殖的影响。结果重组表达质粒pcDNA3.1-JTV1经双酶切及测序证实,目的基因已插入质粒中;人JTV1基因能在K562细胞中稳定表达;JTV1具有抑制K562细胞增殖的作用。结论已成功构建了JTV1基因真核表达质粒,并获得了稳定表达人JTV1基因的K562细胞克隆,为进一步研究人JTV1基因的功能及其与白血病细胞增殖及凋亡的相关性提供了细胞模型。     

Smac-EGFP真核表达质粒的构建

目的构建EGFP融合表达第二线粒体源的半胱氨酸激活物(second mitochondria-derived activator of caspases,Smac)基因的真核表达质粒。方法提取He La细胞总RNA,逆转录合成c DNA,以其为模板,PCR扩增Smac基因,将其插入到p EGFP-N3质粒中,构建重组真核表达质粒p EGFP-N3-Smac,通过脂质体法转染He La细胞,于倒置荧光显微镜下观察转染后质粒在细胞中的分布表达情况,并采用Western blot法鉴定表达的融合蛋白Smac-EGFP。结果成功构建了真核表达质粒p EGFP-N3-Smac,转染后的He La细胞在细胞内膜周围即线粒体部位可见明显、规律的GFP表达;表达的融合蛋白Smac-EGFP相对分子质量约49 000。结论成功构建了重组真核表达质粒p EGFP-N3-Smac,并在He La细胞中表达了融合蛋白,为进一步研究Smac的功能提供了参考。     

单-ADP-核糖基转移酶3过表达对人乳腺癌细胞MDA-MB-231增殖、凋亡及侵袭的影响

目的探讨单-ADP-核糖基转移酶3(mono-ADP-ribosyltransferase 3,ART3)过表达对人乳腺癌MDA-MB-231细胞增殖、凋亡及侵袭的影响。方法在Lipofectamine 2000的介导下,将ART3基因重组真核表达质粒pCMV-ART3-myc-tag转染人乳腺癌细胞MDA-MB-231,用G418进行稳定转染细胞株的筛选,同时设未转染组对照。Western blot法检测稳定转染细胞中ART3-myc-tag蛋白的表达,MTT法、流式细胞术和Transwell试验分别检测ART3对MDA-MB-231细胞增殖活力、凋亡及侵袭能力的影响。结果 ART3可在MDA-MB-231细胞中稳定过表达;ART3转染组细胞的增殖活力和侵袭能力均明显高于未转染组(P0.05);ART3转染组细胞的凋亡率明显低于未转染组(P0.05)。结论过表达ART3能促进乳腺癌细胞增殖、侵袭,并抑制其凋亡,本实验为进一步研究ART3基因在乳腺癌发生发展中的作用及其机制奠定了基础。     

P4HB基因真核表达质粒的构建及在人肺腺癌A549细胞中的表达

目的构建人P4HB基因真核表达质粒,并检测其在人肺腺癌A549细胞中的表达。方法采用RT-PCR法从正常人肝细胞HL7702中扩增P4HB基因片段,插入真核表达载体pcDNA3.1(+),构建重组真核表达质粒pcDNA3.1-P4HB,在脂质体LipofectamineTM 2000介导下转染A549细胞,通过G418加压筛选,建立稳定转染细胞系,通过qPCR和Western blot法检测转染细胞中P4HB基因mRNA的水平及蛋白的表达水平。结果重组真核表达质粒pcDNA3.1-P4HB经双酶切(BamHⅠ/EcoRⅠ)和测序证实构建正确;质粒pcDNA3.1-P4HB转染的A549细胞中P4HB基因mRNA的水平为空载体转染的细胞和未转染细胞的102倍;质粒pcDNA3.1-P4HB转染的A549细胞中P4HB蛋白的表达量(1.71)较未转染的A549细胞(1.11)明显升高。结论成功构建了P4HB基因真核表达质粒pcDNA3.1-P4HB,转染A549细胞后,P4HB基因在mRNA水平和蛋白水平均出现了较强的表达,表明P4HB基因已稳定转染入A549细胞中。本实验为进一步研究P4HB对人肺癌细胞增殖、侵袭和转移能力的影响奠定了基础。     

Lipocalin-2基因表达产物对人胚肾细胞增殖的影响

目的构建Lipocalin-2(Lcn-2)基因真核表达质粒PL-2,并检测其及前期原核表达的重组Lcn-2蛋白对人胚肾细胞HEK293增殖的影响。方法利用RT-PCR从鼠巨噬细胞RAW264.7中扩增Lcn-2基因,克隆入真核表达质粒pEGFP-C1中,构建重组质粒PL-2。转染HEK293细胞,通过荧光观察和RT-PCR鉴定Lcn-2基因的表达。将重组质粒PL-2和前期原核表达的重组Lcn-2蛋白作用于HEK293细胞,通过MTT法检测细胞增殖情况,Westernblot法检测细胞增殖核抗原(PCNA)的表达。结果重组真核表达质粒PL-2经酶切和测序鉴定,证明构建正确。经荧光观察和RT-PCR鉴定,转染重组质粒PL-2的HEK293细胞中有Lcn-2基因的表达。加入重组Lcn-2蛋白后,HEK293细胞较对照组明显增殖,细胞中PCNA的表达也较对照组明显升高,而重组质粒PL-2对HEK293细胞增殖以及细胞中PCNA的表达均无影响。结论已成功构建了Lcn-2基因真核表达质粒,其对HEK293细胞的增殖无影响,而原核表达的重组Lcn-2蛋白对HEK293细胞的增殖有一定的促进作用,推测Lcn-2基因的表达产物可能通过与细胞膜受体结合促进HEK293细胞的增殖。     

小鼠丝氨酸/精氨酸蛋白特异激酶2基因真核重组表达质粒的构建及其对微管蛋白聚合的影响

目的构建小鼠丝氨酸/精氨酸蛋白特异激酶2(serine/arginine-rich protein specific kinase 2,SRPK2)基因(srpk2)的真核重组表达质粒,并分析其对微管蛋白α-Tubulin聚合的影响。方法利用RT-PCR法扩增srpk2的全长cDNA序列,通过分子克隆技术构建真核重组表达质粒pLV-EGFP(2A)Puro-srpk2;在脂质体Lipofectamine 2000的介导下,将真核重组表达质粒及空载体pLV-EGFP2(A)Puro分别转染人胚肾HEK293T细胞,同时设空白对照组(未转染)。采用RT-PCR及Western blot法分别检测HEK293T细胞中srpk2基因mRNA转录及SRPK2蛋白的表达情况;Western blot法分析HEK293T细胞中聚合态和游离态α-Tubulin的含量。结果双酶切及DNA序列鉴定证明真核重组表达质粒pLV-EGFP2(A)Puro-srpk2构建正确,且在HEK293T细胞中实现了srpk2基因过表达。真核重组表达质粒转染组HEK293T细胞中游离态α-Tubulin含量显著低于空载体转染组及空白对照组(P0.05),而聚合态α-Tubulin水平明显高于空载体转染组及空白对照组(P0.05)。结论成功构建了srpk2基因的真核重组表达质粒,SRPK2可促进α-Tubulin微管蛋白聚合。     

双启动子双报告基因真核表达质粒的构建及鉴定

目的构建双启动子双报告基因真核表达质粒,并在293T细胞中表达,为筛选携带报告基因的肿瘤细胞系提供分子工具。方法以质粒pcDNA3. 1、pcDNA6-GFP、pSVA-Luc为模板,分别扩增CMV、GFP和Luc基因序列,通过In-fusion法将3个基因片段与经HindⅢ、XhoⅠ双酶切的pcDNA3. 1载体连接,构建双启动子双报告基因重组真核表达质粒pC-T2A-Luc-GFP,转染293T细胞,荧光显微镜直接观察GFP的表达,化学发光仪检测Luc的表达。提取293T细胞总RNA,RT-PCR法扩增p53基因,通过In-fusion法将p53基因连接于质粒pC-T2A-Luc-GFP上,构建质粒pC-p53-T2A-Luc-GFP,转染293T细胞,Western blot法检测p53的表达,化学发光仪检测Luc的表达。结果重组真核表达质粒pC-T2A-Luc-GFP经酶切及测序鉴定构建正确,GFP和Luc双报告基因均可在293T细胞中正确表达,且可用于后续的动物模型建立。pC-p53-T2A-Luc-GFP质粒中p53的插入降低了Luc的表达,但其表达仍为阳性,不影响Luc的正常检测。结论成功构建了双启动子双报告基因真核表达质粒pC-T2A-Luc-GFP,并可在293T细胞中表达,为建立携带报告基因的肿瘤细胞系及肿瘤模型奠定了分子基础。     

FOXC1蛋白基因shRNA干扰质粒对胃癌SGC-7901细胞增殖的影响

目的探讨FOXC1蛋白基因RNA干扰质粒对胃癌SGC-7901细胞增殖的影响。方法采用RT-PCR及Westernblot法检测SGC-7901细胞和胃黏膜上皮细胞GES-1中FOXC1基因mRNA的转录水平和蛋白的表达水平。构建3个FOXC1基因shRNA真核表达质粒pshRNA-FOXC1a、pshRNA-FOXC1b、pshRNA-FOXC1c,分别转染SGC-7901细胞,并设pGenesil-1空质粒转染组和未转染的细胞对照组,RT-PCR及Western blot法分别检测转染细胞中FOXC1基因mRNA的转录水平和蛋白的表达水平;MTT法检测转染细胞的增殖活力;流式细胞术检测转染细胞的细胞周期。结果 SGC-7901细胞中FOXC1基因mRNA的转录水平及蛋白的表达水平均显著高于GES-1细胞(P<0.05)。pshRNA-FOXC1a组、pshRNA-FOXC1b组和pshRNA-FOXC1c组SGC-7901细胞中FOXC1基因mRNA的转录水平和蛋白的表达水平均显著低于pGenesil-1组和SGC-7901组(P<0.01);细胞增殖抑制率显著高于pGensil-1组(P<0.05)。处于G1、G2期的细胞比例显著高于SGC-7901组(P<0.01),S期细胞比例显著低于SGC-7901组(P<0.01)。结论成功构建了FOXC1蛋白基因shRNA真核表达质粒。FOXC1在胃癌细胞SGC-7901中呈高表达,抑制其表达后,细胞周期被阻滞在G1～G2期,细胞增殖受到抑制。     

人NIRF基因真核表达质粒的构建及其在HepG2.2.15细胞中的表达

目的构建人NIRF基因真核表达质粒,并在HepG2.2.15细胞中表达NIRF蛋白。方法从HeLa细胞中提取总RNA,设计特异性引物,通过RT-PCR法扩增NIRF基因编码区全长序列,插入到pIRES2-EGFP真核表达载体中,构建重组真核表达质粒pIRES2-EGFP-NIRF,转染HepG2.2.15细胞,检测细胞中NIRF基因mRNA的转录水平及蛋白的表达水平。结果重组真核表达质粒pIRES2-EGFP-NIRF经双酶切及测序鉴定证明构建正确,转染HepG2.2.15细胞后,可检测到细胞中NIRF基因mRNA的转录及蛋白的表达。结论已成功构建了人NIRF基因真核表达质粒,并在HepG2.2.15细胞中表达了NIRF蛋白,为下一步研究其在肿瘤组织中的功能奠定了基础。     

乙苯,苯乙烯的生产及合成乙苯,苯乙烯催化剂的研究进展

苯乙烯是重要的石油化工原料。本文介绍了国内外苯乙烯生产的发展概况，合成乙苯，苯乙烯所用的催化剂种类，并就苯乙烯生产及催化剂的发展趋势提出了自己的观点。     

Synthesis and characterization of homopolymers and copolymers of various acrylates and acrylonitrile

Various homopolymers and copolymers of methyl acrylate, ethyl acrylate, butyl acrylate, and acrylonitrile in different feed ratios were synthesized. These were characterized by IR, ¹³C-NMR, DSC, DTA, and TGA. Spectroscopic characterization helped in differentiating copolymers of different mol ratios. Thermal analysis revealed different degradation patterns for homopolymers and copolymers. The temperature and energy changes associated with various phase transitions were dependent on the chemical composition of homo- and copolymers, as expected. © 1993 John Wiley & Sons, Inc.     

生物质气化及生物质与煤共气化技术的研发与应用

总结了生物质原料的特点及生物质单独气化的缺点;介绍了国内外生物质气化技术及生物质与煤共气化技术的研发与应用现状;分析了在此领域国内外的发展趋势与前景;概括了开展生物质与煤共气化技术研发的意义。     

Electronic structures and reactivities of monometallic and bimetallic clusters of Au and Cu

The variation of the Au 4f binding energy of Au clusters with the cluster size has been established by measuring the binding energies of clusters whose size distributions were independently determined by HREM and STM. The binding energy increases significantly when the cluster size is less than 2 nm. Au-Cu bimetallic clusters of the composition Cu₃Au have been deposited for the first time on carbon substrates. The shifts in the core level binding energies of the bimetallic clusters show the effect of alloying in the case of large clusters, but show effects of both alloying and cluster size in the case of the small clusters. The interaction of CO with Cu₃Au clusters is stronger than with a bulk Cu metal. The interaction of CO with small Cu clusters also seems to be stronger than with bulk Cu or with large Cu clusters.     

钾盐资源及钾肥供需情况分析及预测

阐述了国内外钾盐资源及钾肥生产现状,对国内外钾盐的供需形势进行了分析及预测,从资源、原材料、国际市场三方面提出了解决我国钾盐短缺的措施。     

Kinetics and Mechanism of Oxidation of Mono- and Polyacetals

Abstract

Kinetics and mechanisms of oxidation of 6 acetals by molecular oxygen and ozone in liquid phase have been studied. Reaction with molecular oxygen (70°C, 15–16 hr) leads to the formation monoethers of the corresponding glycols with 68–90% selectivity. Salts of metals and complexes with crown-ethers have increased the reaction rate significally. Ozone have reacted with acetals with formation similar products. The mechanisms of intermediate stages have been proposed.     

Comparison of the Adhesion and Cohesion of Triblock and Random Copolymers of Styrene and Butadiene

A substantially greater detachment energy is required to strip a polyethylene tereph-thalate (Mylar) film from a styrene-butadiene-styrene (SBS) triblock copolymer compared to that for peeling from a random styrene-butadiene (SBR) copolymer. This is true even though the intrinsic interaction between the Mylar and each elastomer is expected to be similar because of their virtually identical chemical composition. It is proposed that this difference in peel strength (between the SBS and SBR) is a consequence of the much higher dissipative capacity of the former elastomer. Another manifestation of this is the higher cohesive tear strength of the SBS compared to the SBR. Extents of energy dissipation within each elastomer during detachment of the Mylar adherend are consistent with the hypothesis that the average maximum stress experience before detachment is some similar fraction of each elastomer's tensile strength.     

Wetting properties of homopolymers and copolymers of pentafluorostyrene and methylacrylate and homopolymer blends

Polypentafluorostyrene (PPFS), polymethylacrylate (PMA), and poly(pentafluorostyrene-co-methylacrylate), poly(PFS-co-MA) were prepared and the wetting characteristics of polymer blends of PPFS and PMA were compared with that of poly(PFS-co-MA) via contact angle measurements. The critical surface tension of polypentafluorostyrene was found to be 22.6 dyne/cm, which is comparable to the value reported for polytrifluoroethylene (22 dyne/cm). The critical surface tension of poly(PFS-co-MA) is not linearly related to its composition. The polymer blends of PPFS and PMA exhibit significant surface enrichment of the fluoropolymer. The harmonic-mean method¹ was employed to determine surface tensions of these polymers and many known polymers. It is found that the method produces useful surface tension data provided the contact angle values are derived from testing liquids of dissimilar polarity.     

实施水泵节能技改 实现节能降耗

介绍了内蒙古亿利化学工业有限公司在满足生产的前提下,对现有的循环水泵实施的节能技改及效果。     

责任和责任心的涵义与结构

责任是人应主动承担的角色义务和对其因过失所造成后果应承担的责罚.有两层涵义:一是义务;二是后果.责任心是个体自觉做好分内事务和履行道德义务的心理倾向,是个性心理品质成分中自我特征维度上的重要内容.责任心具有两个方面的涵义:一是角色分内职责;二是角色道德义务.责任心是一种通过责任认知、责任个性和责任适应的动态形式表现出来的静态品质,责任心是责任心过程结构与责任心关系结构相互制约、相互影响的统一体.