基于Cre／Loxp系统Tyr03、Axl受体酪氨酸激酶转基因小鼠的建立

采用大肠杆菌β一半乳糖酶基因LacZ作为报告基因,建立Tyr03、Axl受体酪氨酸激酶含有loxp位点的转基因小鼠。与不同组织特异性Cre小鼠杂交,获得时空性表达Tyr03、Axl的转基因小鼠,为研究其在各个组织中的功能奠定基础。方法构建含有loxp位点的Tyr03、Axl受体酪氨酸激酶质粒,测序正确后,通过显微注射法将插入CAG启动子下游的Geo—STOP—msAxl及Geo—STOP—msTyr03基因的转基因载体注射入BDFI供体鼠受精卵,移植受精卵至ICR受体鼠,待产仔后,通过双引物PCR鉴定F。以及Fn代小鼠的基因型,RT—PCR,X—gal染色观察小鼠组织中LaeZ基因,运用WB（WesternBlot）检测转基因小鼠和野生型小鼠体内目的蛋白表达的差异。结果获得了含有目的基因的转基因阳性小鼠;通过RT—PCR对阳性小鼠证明LacZ基因的存在;对存在LacZ基因的小鼠进行X—gal染色,在Geo—STOP—msTyr03的脑、睾丸、胸腺中观察到蓝色沉淀,在Geo—STOP—msAxl的脑、心、肾中观察到蓝色沉淀,间接说明目的基因能表达的组织和器官;对双阳性的Geo．STOP．msTyr03、Geo．STOP—msAxl转基因小鼠和野生型小鼠,WB检验证实Tyr03、Axl基因的表达没有差异,说明在加入stop序列之后,Tyr03基因确实存在,而且表达受到抑制。结论成功建立Geo-STOP—msAxl及Geo—STOP—msTyr03酪氨酸受体转基因小鼠。     

β—半乳糖苷酶基因在小鼠乳腺细胞表达的研究

构建了以乳清酸蛋白（ＷＡＰ）５′区２．６ｋｂ为调控序列,指导β－半乳糖苷酶基因的真核表达质粒,采用直接注射质粒于乳腺的方法在小鼠乳腺中表达出β－半乳糖苷酶活性,证实ＷＡＰ基因调控序列的功能正确性,可以用于转基因动物乳腺表达研究,同时证实该方法可以作为一种暂时性表达载体的验证方法．     

以淀粉酶基因为筛选标记的E.coli克隆载体的构建

以E．coli质粒pJRD184为基础,将多酶切点引入地衣芽胞杆菌（Bacilluslicheniformis）的a－淀粉酶基因信号肽编码区的PstI切点,构建了能表达并将a－淀粉酶（Amy）渗漏到胞外的E．coli克隆载体pLAM9712．该载体可根据在含有可溶性淀粉的LB平板上菌落周围有没有淀粉水解圈直接筛选重组子,从而避免了使用异丙基硫代－β－D－半乳糖苷（IPTG）、5－溴－4－氯－3－吲哚－β－D一半乳糖苷（X－gal）,代之以廉价的碘、淀粉．实验进一步验证了pLAM9712在基因克隆中的可行性及质粒本身的稳定性．     

HIF-1调控小鼠bace1基因的转录表达

阿尔茨海默症的一个关键致病原因是大脑中淀粉样蛋白（A13）的过度产生或堆积．Aβ是由其前体蛋白APP经β-和γ-分泌酶依次水解而生成的，但对于这些分泌酶基因表达的转录调控的了解还很少．由于大脑发生缺氧／缺血会造成Aβ的产量增加，而缺氧时所激活的转录因子HIF-1（Hypoxia-Inducible Factor 1）会调控下游多种基因的表达，我们对HIF-1是否参与调控β-分泌酶的表达进行了研究．对小鼠β-分泌酶基因bace1的调控序列进行分析，发现其中含有一个缺氧应答元件（Hypoxia-Responsive Element，HRE）．我们的数据显示，HRE突变片段启动报告基因荧光素酶表达的活性比正常序列片段的启动活性明显降低．电泳迁移率的变动分析（EMSA）实验进一步证实，HIF-1可以与小鼠bace1中HRE元件相互作用．当在哺乳动物细胞中过度表达HIF-1时，BACE1的mRNA水平和蛋白质水平均有明显的增高．这些结果表明小鼠bace1基因的表达受转录因子HIF-1的调控．鉴于目前阿尔茨海默症研究领域都把抑制BACE1作为首选治疗靶位，HIF-1因而有可能成为治疗AD的一种药物靶点．     

利用目的基因转化技术培育棉花抗病新品种

用花粉管通道法将菜豆几丁质酶基因与烟草β－1、3葡聚糖酶基因双价植物表达载体pBLGC导入新疆棉花主栽品种（系）系550、822、1304、石远321。通过卡那霉素检测、PCR－Southern验证、抗黄萎病、枯萎病性筛选，得到抗病性良好且遗传性稳定的转基因棉花。经过6年10个世代的选育，育成抗枯萎病、抗（耐）黄萎病的转基因抗病新品系。     

人凝血Ⅸ因子乳腺特异性表达载体在离体细胞和小鼠乳腺组织中表达的初步研究

以小鼠MAR（matrixattachmentregion）元件,牛β－酪蛋白（β－casein）基因5＇端2．0kb调控顺序,人凝血Ⅸ因子小基因（hFIXminigene）构建乳腺组织特异性表达载体,表达载体和质粒pSV－neo共转染中国仓鼠卵巢细胞（CHO）,兔皮肤纤维细胞（RSF）和人胚肾上皮细胞（293细胞）．发现hFIX基因在CHO细胞中获得低水平表达,最高表达量为7．2ng／ml．在皮肤成纤维细胞中的表达量低于2ng／ml．在293细胞中没有表达．表达载体用stearylamine（SA）脂质体包埋后尾静脉注射哺乳期小鼠,hFIX蛋白在小鼠乳汁中表达水平高达87．34ng／ml．     

用慢病毒载体介导产生绿色荧光蛋白（GFP）转基因小鼠

以慢病毒（lentivirus）载体为骨架，携带绿色荧光蛋白（GFP）基因的假病毒，通过小鼠受精卵卵周隙注射，将其感染小鼠受精卵，经移植于假孕母鼠后获得转基因小鼠．应用PCR、荧光显微镜观察和流式细胞仪分析等技术，证明了GFP基因的整合率达到40%以上；实时定量PCR分析结果表明转基因小鼠中GFP基因整合的拷贝数约为40；染色体荧光原位杂交分析结果显示GFP基因在小鼠染色体上的整合是随机的，并通过交配可将外源基因遗传至子代，获得的多整合位点和不同表达水平的转基因小鼠具有明显的实际应用和研究价值．文中报道的慢病毒载体介导的转基因技术为高效制备和选育高表达转基因小鼠品系提供了一种有效的途径．     

迷走神经在心室颤动发生中的作用

在小鼠吸入氯仿诱发心室颤动（ＶＦ）的模型中，毒扁豆碱（０．５ｍｇ／ｋｇ）或氯化乙酰－β－甲基胆碱（０．６ｍｇ／ｋｇ）不能防止心室颤动发生．利血平（１ｍｇ／ｋｇ，５ｈ）耗竭心脏儿茶酚胺（ＣＡ）或摘除两侧肾上腺以降低小鼠的儿茶酚胺总水平后，虽然氯仿仍诱发心室颤动，但毒扁豆碱（０．１５或０．２ｍｇ／ｋｇ）或氯化乙酰－β－甲基胆碱（０．４ｍｇ／ｋｇ）都有明显的防颤作用．阿托品（３ｍｇ／ｋｇ）可阻断毒扁豆碱对利血平化小鼠的防颤作用．在正常小鼠中，毒扁豆碱（０．１５ｍｇ／ｋｇ）与氯化乙酰－β－甲基胆碱（０．４５ｍｇ／ｋｇ）复合作用亦可防止心室颤动发生．结果显示：在正常小鼠中，快速吸入氯仿通过激动交感神经引起内源性儿茶酚胺释放诱发心室颤动的过程中，迷走神经释放的乙酰胆碱，则在心室颤动发生上起对抗作用，但尚不足以抑制心室颤动发生．     

AVP对VSMC脂质过氧化的影响及CGRP、SP的调节作用

目的：研究AVP对VSMC脂质过氧化的影响及CGRP、SP的调节作用。方法：以培养的大鼠血管平滑肌细胞（VSMC）为模型,动态观察了精氨酸加压素（AVP）对VSMC脂质过氧化的影响,及降钙素基因相关肽（CGRP）、P物质（SP）对AVP作用的调节,旨在探讨它们在高血压病发病中的意义。结果：（1）10－7M的AVP作用后,VSMP的丙二醛（MDA）含量明显高于对照组（P＜0．01）;（2）10－7M的CGRP、SP分别与10－7M的AVP共同作用后,VSMP的MDA含量均减少,与AVP对照组比较,差异非常显著（P＜0．01）。结论：AVP参与高血压病的发病与其致VSMC的脂质过氧化损伤有关,CGRP、SP对其有拮抗作用。     

Markedness and its Interpretation in Language Use

Language markedness is a common phenomenon in languages, and is reflected from hearing, vision and sense, i.e. the variation in the three aspects such as phonology, morphology and semantics. This paper focuses on the interpretation of markedness in language use following the three perspectives, i.e. pragmatic interpretation, psychological interpretation and cognitive interpretation, with an aim to define the function of markedness.     

Features of Women's language

Language is a means of verbal communication. People use language to communicate with each other. In the society, no two speakers are exactly alike in the way of speaking. Some differences are due to age, gender, statue and personality. Above all, gender is one of the obvious reasons. The writer of this paper tries to describe the features of women＇s language from these perspectives： pronunciation, intonation, diction, subjects, grammar and discourse. From the discussion of the features of women＇s language, more attention should be paid to language use in social context. What＇s more, the linguistic phenomena in a speaking community can be understood more thoroughly.     

低渗透非均质砂岩油藏启动压力梯度研究

理论推导与室内实验相结合,建立了低渗透非均质砂岩油藏启动压力梯度确定方法。首先借助油藏流场与电场相似的原理,推导了非均质砂岩油藏启动压力梯度计算公式。其次基于稳定流实验方法,建立了非均质砂岩油藏启动压力梯度测试方法。结果表明:低渗透非均质砂岩油藏的启动压力梯度确定遵循两个等效原则。平面非均质油藏的启动压力梯度等于各级渗透率段的启动压力梯度关于长度的加权平均;纵向非均质油藏的启动压力梯度等于各渗透率层的启动压力梯度关于渗透率与渗流面积乘积的加权平均。研究成果可用于有效指导低渗透非均质砂岩油藏的合理井距确定,促进该类油藏的高效开发。     

On Tragic Consciousness In The Works By Ernest Hemingway

As an American modern novelist who were famous in the literary world, Hemingway was not a person who always followed the trend but a sharp observer. At the same time, he was a tragedy maestro, he paid great attention on existence, fate and end-result. The dramatis personae's tragedy of his works was an extreme limit by all means tragedy on the meaning of fearless challenge that failed. The beauty of tragedy was not produced on the destruction of life, but now this kind of value was in the impact activity. They performed for the reader about the tragedy on challenging for the limit and the death.     

Achieving Unusual States of Matter Under High Pressure

正The periodicity of the elements and the non-reactivity of the inner-shell electrons are two related principles of chemistry,rooted in the atomic shell structure.Within compounds,Group I elements,for example,invariably assume the+1 oxidation state,and their chemical properties differ completely from those of the p-block elements.These general rules govern our understanding of chemical structures and reactions.Using first principles calcula-     

Exchange-Correlation and Electronic Excitation Energies from Pairing Matrix Fluctuations

We have developed an adiabatic connection to formulate the ground-state exchange-correlation energy in terms of pairing matrix linear fluctuations.This formulation of the exchange-correlation energy opens a new channel for density functional approximations based on the many-body perturbation theory.We illustrate the potential of such approaches with an approximation based on the particle-particle Random Phase Approximation(pp-RPA).This re-     

Density Functional Theory for the Response of Periodic Systems to Electric Fields Based on the Vector Potential Approach

正The electronic and nuclear(structural/vibrational)response of 1D-3D nanoscale systems to electric fields gives rise to a host of optical,mechanical,spectral,etc.properties that are of high theoretical and applied interest.Due to the computational difficulty of treating such large systems it is convenient to model them as infinite and periodic(at least,in first approximation).The fundamental theoretical/computational problem in doing so is that     

On the Thermodynamic Limit for Responses to Static Electromagnetic Fields

For molecular systems,the quantum-mechanical treatment of their responses to static electromagnetic fields usually employs a scalar-potential treatment of the electric field and a vector-potential treatment of the magnetic field.Although the potential for each field separately is associated with the choice of an(unphysical)origin,the precise choice of the origin for the electrostatic field has little consequences for the results.This is different for the     

Call for Papers

<正>"The Journal of Shanghai Normal University:Mathematics"is published by Shanghai Normal University as regular issues of The Journal of Shanghai Normal University each year from 2014 in English.The editors-in-chief of the issues are professors Yuhao Cong and Maoan Han.The Journal of Shanghai Normal University was started in 1958 with     

Journal of Donghua University ( English Edition) Contents of Volume 31

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