FAM92A1-289基因的真核表达载体构建和亚细胞定位

利用PCR方法扩增FAM92A1-289全长,经BamH I和Xho I酶切后连接入pEGFP-N1真核表达载体,构建pEGFP-N1-FAM92A1-289重组表达质粒,转染Hela细胞,利用荧光显微镜观察FAM92A1-289在细胞中的定位。经双酶切和核酸序列分析证实重组质粒包含有正确编码的FAM92A1-289读码框。荧光显微镜观察到空质粒pEGFP-N1转染后,整个细胞内弥散绿色荧光,而转染pEGFP-N1-FAM92A1-289重组载体后,可见绿色荧光分布于Hela细胞核中,显示FAM92A1-289定位于细胞核。成功构建人FAM92A1-289真核表达载体,FAM92A1-289定位于哺乳细胞的细胞核中。     

九香虫血淋巴对肿瘤细胞凋亡相关蛋白表达的影响

探索九香虫血淋巴诱导肿瘤细胞凋亡的作用通路。利用Bradford法检测九香虫血淋巴浓度并将其作用于体外培养的人乳腺癌MCF-7细胞、人胃癌SGC-7901细胞,Western blot法检测经九香虫血淋巴干预后肿瘤细胞凋亡相关蛋白Caspase-3、Caspase-8、Caspase-9、Bcl-2、Bax等的表达。结果显示,九香虫血淋巴作用的SGC-7901、MCF-7细胞中Caspase-3、Caspase-9、Bax蛋白的表达较对照组细胞明显上调;两种细胞的Bcl-2蛋白,较对照组细胞表达明显下调;两种细胞的Caspase-8蛋白,较对照组细胞表达无明显差异。结果表明,经九香虫血淋巴诱导的SGC-7901、MCF-7细胞可能通过触发其线粒体凋亡途径使肿瘤细胞发生不可逆的凋亡。     

Snai1 miRNA干扰质粒构建及胃癌细胞系稳定株筛选

目的：设计并构建针对Snai1的微小干扰核糖核酸（miRNA）,最终鉴定出有效干扰质粒并筛选稳定转染的胃癌细胞株SGC-7901。方法：设计并构建4对Snai1的pcDNATM6.2-GW/EmGFPmiR microRNA及1对无效对照microRNA干扰质粒。将干扰质粒用罗氏BD转染试剂转染胃癌细胞株SGC-7901,通过倒置荧光显微镜观察绿色荧光确定转染效率。分别用不同浓度l的杀稻瘟菌素作用于SGC-7901细胞,得到杀稻瘟菌素对SGC-7901细胞的筛选浓度。Westernblot检测4对干扰质粒、阴性对照质粒对snai1蛋白水平表达的影响。结果：测序表明,Snai1干扰序列及读码框完全正确,干扰质粒瞬时转染的SGC-7901细胞系在倒置荧光显微镜下观察绿色荧光达85%以上。杀稻瘟菌素对于SGC-7901细胞的筛选浓度为5μg/ml。Westernblot结果显示,干扰序列Mi-1对Snai1有较强的干扰效果。结论：成功构建了Snai1干扰真核表达载体,同时筛选出有效干扰质粒及稳定转染株,为进一步研究Snai1在胃癌中的作用奠定了基础。     

Rab GDP解离抑制因子的定位和表达

目的:构建含人GDI1和GDI2基因的真核表达载体进行定位和蛋白表达研究。方法:用PCR从U251细胞cDNA克隆GDI1和GDI2基因,构建真核表达载体pEGFP-N2-GDI1和pEGFP-N2-GDI2,转染HEK293T细胞。荧光显微镜观察GDI1和GDI2蛋白的细胞内定位,再通过SDS-PAGE和Western blotting鉴定蛋白的表达。结果:成功克隆到1 341bp和1 335bp的人源GDI1和GDI2基因,并准确插入真核表达载体pEGFP-N2中,荧光观察这两个蛋白定位到细胞浆中,并能利用标签抗体检测到GDI1和GDI2的表达。结论:GDI1和GDI2能够定位到细胞浆,并能通过Western blotting检测,为进一步研究GDI1和GDI2的功能奠定了基础。     

人酸性调宁蛋白calponin-3与EGFP的融合表达及其细胞定位

调宁蛋白家族进化保守,广泛分布于肌细胞与非肌细胞。目前发现该蛋白家族有碱性、中性和酸性3个异构体,它们在非肌细胞中的功能尚不明确,而酸性调宁蛋白calponin-3是被研究最少的家族成员。本研究通过RT-PCR的方法从人支气管上皮细胞HBE中克隆编码calponin-3蛋白的CNN3基因,克隆至真核表达载体pEGFP-N1。重组质粒pEGFP-N1-CNN3经酶切鉴定和DNA测序正确后,用于瞬时转染细胞。Western blotting检测293T,A549和SMMC-7721细胞内重组绿色荧光融合calponin-3蛋白的瞬时表达,激光共聚焦显微镜分析calponin-3-绿色荧光融合蛋白与罗丹明-鬼笔环肽标记F-actin的细胞定位。结果表明成功构建了真核表达载体pEGFP-N1-CNN3并在293T、A549和SMMC-7721细胞中高效表达;通过绿色荧光结合免疫荧光染色确定calponin-3与F-actin共定位。成功构建calponin-3蛋白的表达载体,在细胞中瞬时表达并检测其细胞亚定位,为进一步研究calponin-3蛋白的生物学功能奠定基础。     

人星状病毒非结构蛋白基因nsP1a/1真核表达载体构建及其

目的 将人星状病毒非结构蛋白nsP1 a./1基因连接到真核表达载体上,转染人胚肾上皮细胞48 h后检测其表达.方法 设计特异性引物PCR扩增人星状病毒非结构蛋白nsP1 a/1片段,分别插入真核表达载体pcDNA3.1(+)和pEGFP-N2载体,构建重组表达质粒pcDNA3.1(+)-nsP1a/1-His和pEGFP-N2-nsP1a/1.在转染试剂PEI的介导下将重组表达质粒分别转染293T细胞,转染48 h后分别在荧光显微镜下观察EGFP的表达以及通过Western blot检测nsP1a/1基因的表达.结果 重组表达质粒pcDNA3.1(+)-nsP1a/1-His和pEGFP-N2-nsP1a/1构建成功;转染pEGFP-N2-nsP1a/1后48 h能够在荧光显微镜蓝色激发光下观察到较强的黄绿色荧光;转染pcDNA3.1(+)-nsP1a/1-His后48 h收集细胞进行Western blot检测,能够检测到nsP1a/1-His融合报告基因的表达.结论 成功构建了人星状病毒非结构蛋白nsP1a/1基因真核表达质粒,并在人胚肾上皮细胞293T细胞获得表达,为进一步深入研究nsP1a/1在人星状病毒抵御宿主细胞抗病毒天然免疫中是否发挥作用奠定了基础.     

人Tim-3 基因真核表达载体的构建及表达

目的:为了构建可在真核细胞中高效表达人Tim-3(hTim-3)的真核表达质粒,以便用于hTim-3肿瘤免疫治疗研究。方法:取健康人外周血的单个核细胞,用高保真聚合酶扩增hTim-3基因,先进行hTim-3基因的亚克隆,用BglⅡ和SalⅠ限制性内切酶切下带有酶切位点的hTim-3基因,最终构建hTim-3基因的真核表达质粒pEGFP-N1-hTim-3。用脂质体方法转染质粒至肝癌细胞SMMC7721和巨噬细胞U937中。48h后荧光显微镜观察转染细胞绿色荧光表达情况,初步判断转染效率。结果:酶切和测序鉴定证实目的基因hTim-3正确插入到真核表达载体pEGFP-N1中,转染肝癌细胞SMMC7721和巨噬细胞U937后,在荧光显微镜下观察到绿色荧光蛋白的表达。结论:成功构建了可在真核细胞中高效表达hTim-3基因的真核表达质粒pEGFP-N1-hTim-3,为进一步研究hTim-3的肿瘤免疫治疗奠定了基础。     

大鼠大麻素Ⅰ型受体绿色荧光融合蛋白真核表达载体的构建与鉴定

目的:构建大鼠大麻素型Ⅰ受体绿色荧光融合蛋白真核表达载体并观察其在细胞中的表达。方法:大鼠CB1基因序列设计引物,以大鼠脑组织为模板扩增CB1基因编码区片段,克隆至增强型绿色荧光蛋白表达载体pEGFP-N3中,构建重组融合蛋白表达载体pCB1-EGFP。将pCB1-EGFP质粒转染HeLa细胞,通过观察EGFP报告基因的表达以及免疫荧光,Western Blot方法鉴定CB1可在真核细胞中过表达情况。结果:构建重组融合蛋白表达载体pCB1-EGFP,单双酶切和测序验证正确。将pCB1-EGFP质粒转染HeLa细胞,荧光显微镜下观察到融合表达的绿色荧光蛋白,且呈胞膜表达。免疫荧光试验也证明重组载体转染后,CB1基因和GFP共同定位于胞膜部分。Western Blot实验证明表达CB1蛋白。结论:成功构建了高表达的CB1-EGFP融合蛋白真核表达载体。     

小鼠Myf5真核表达载体的构建及其在细胞中的定位

目的：获得小鼠生肌调节因子Myf5基因并构建pEYFP-C1真核表达载体,观察Myf5在小鼠C3H10T1/2细胞中的定位。方法：利用PCR获得Myf5基因克隆到pEYFP-C1载体中,利用脂质体将构建的表达载体转染C3H10T1/2细胞,荧光观察融合蛋白的表达。结果：从小鼠cDNA文库中得到760bp的myf5的CDS序列后,重组到pEYFP-C1载体中并转染C3H10T1/2细胞,荧光显示Myf5蛋白定位在细胞核中。结论：Myf5载体成功构建并在小鼠C3H10T1/2细胞表达,证明了Myf5蛋白定位于细胞核,为进一步研究Myf5与其他蛋白的相互作用奠定了基础。     

Embryo and Endosperm Function and Failure inSolanumSpecies and Hybrids

Although the importance of the endosperm as a food store inmany angiosperm seeds is well known, its significance duringearly embryogenesis has been neglected. In many interspecifichybrids, and in some other situations, embryos do not developfully and abort. It has often been stated that this is causedby the endosperm failing to conduct sufficient nutrients tothe embryo, but seldom has it been suggested that the endospermactively controls most of the early stages of morphogenesisof the embryo. Information gleaned from a broad survey of theliterature, combined with additional evidence presented here,obtained fromSolanum incanumand interspecific hybrids, indicatethat the endosperm is dynamic and very active in regulatingearly embryo development. This requires highly integrated geneticcontrol of rapidly changing metabolism in the endosperm. Ininterspecific hybrids, lack of coordination may cause unbalancedproduction of growth regulating substances by the endospermand hence abortion of the embryo, or even unregulated productionof nucleases and proteases resulting firstly in autolysis ofthe endosperm and then digestion of the embryo. The endospermmay thus serve to detect inappropriate hybridization of speciesor ploidy levels and so prevent waste of resources by producingseeds that would result in sterile hybrids or unthrifty subsequentgenerations. This discriminatory function of the endosperm hasdiminished during evolution and domestication of the crop plantSolanummelongenaL.Copyright 1998 Annals of Botany Company Solanum, embryo morphogenesis, endosperm, hybrid, seed development.     

环腺苷酸应答元件结合蛋白与学习记忆

环腺苷酸(cAMP)应答元件结合蛋白(cAMP response element binding protein,CREB)是一种核转录因子,可与cAMP反应元件结合,调节基因转录,具有调节精子生成,昼夜节律,学习记忆等功能.近年来关于其在学习记忆中的作用成为医学研究热点.CREB是神经元内多条信息传递途径的汇聚点,参与长时记忆形成和突触可塑性.长时记忆(long-term memory)形成需依赖CREB介导的基因转录,干扰或抑制CREB活性可破坏长时记忆.长时程增强(long-term potentiation,LTP)是研究学习记忆的理想模型,在LTP诱导和维持过程中均可观察到CREB活性持续升高.但增龄过程中,海马CREB活性下降,影响学习记忆功能,与许多神经退行性疾病发生有关.     

Pragmatic Women and Body Politics and Maternities and Modernities: Colonial and Postcolonial Experiences in Asia and the Pacific

Pragmatic Women and Body Politics. Margaret Lock and Patricia A. Kaufert. eds. Cambridge, UK: Cambridge University Press, 1998. xii +364 pp.
Maternities and Modernities: Colonial and Postcolonial Experiences in Asia and the Pacific. Kalpana Ram and Margaret Jolly. eds. Cambridge, UK: Cambridge University Press, 1998. xiv. 305 pp.     

HIFs and tumors--causes and consequences

For most organisms oxygen is essential fo life. When oxygen levels drop below those required to maintain the minimum physiological oxygen requirement of an organism or tissue it is termed hypoxia. To counter act possible deleterious effects of such a state, an immediate molecular response is initiated causing adaptation responses aimed at cell survival. This response is mediated by the hypoxia-inducible factor-1 (HIF-1), which is a heterodimer consisting of an alpha- and a beta-subunit. HIF-1 alpha protein is stabilized under hypoxic conditions and therefore confers selectivity to this response. Hypoxia is characteristic of tumors, mainly because of impaired blood supply resulting from abnormal growth. Over the past few years enormous progress has been made in the attempt to understand how the activation of the physiological response to hypoxia influences neoplastic growth. In this review some aspects of HIF-1 pathway activation in tumors and the consequences for pathophysiology and treatment of neoplasia are discussed.