重组GATA4腺病毒的构建及心肌细胞感染

利用AdEasy腺病毒表达系统构建了GATA4基因过表达腺病毒.编码大鼠GATA4基因的目的片段克隆入pAdTrackcmv质粒,pAdTtrackcmv-GATA4穿梭质粒经PmeⅠ线性化后转入含pAdEasy-1的BJ5183菌进行同源重组.pAdEsay-GATA4重组质粒经卡那霉素抗性筛选及PacⅠ酶切鉴定.pAdEsay-GATA4转入293A细胞进行包装,产生具有感染性的重组病毒.Ad-GATA4重组病毒感染HeLa及乳鼠心肌细胞,通过免疫印迹及实时定量聚合酶链反应(Real-time PCR)检测GATA4表达及促心肌肥大效应.Ad-GATA4重组病毒感染乳鼠心肌细胞后,诱导心肌细胞表面积明显增加,ANF表达明显增强.结果表明,Ad-GATA4腺病毒成功感染心肌细胞并诱导了大鼠心肌肥大表型的出现.     

腺病毒介导的DPP6过表达载体的构建及其在大鼠心肌细胞内表达的鉴定

目的:构建腺病毒介导的DPP6过表达载体并验证其在大鼠心肌细胞内的表达.方法:应用PCR技术扩增目的基因DPP6,经酶切、连接等反应将目的基因插入穿梭载体p Shuttle-CMV中,进而转化至含有Ad Easy质粒的大肠杆菌中,进行重组;所产生的腺病毒载体转染在HEK-293细胞中,进行腺病毒的包装、分泌和扩增,所得病毒感染大鼠心肌细胞,进行荧光及实时定量PCR(q PCR)鉴定.结果:DPP6过表达腺病毒载体构建成功,经HEK293细胞包装、扩增后,所得病毒可成功感染初生大鼠心肌细胞,观测到腺病毒携带的绿色荧光蛋白表达,q PCR检测DPP6 mRNA表达明显升高.结论:成功构建了腺病毒介导的DPP6过表达载体,并在大鼠心肌细胞内成功表达,为进一步研究DPP6在心肌细胞内的功能奠定了基础.     

腺病毒介导ERK-2基因在生长板软骨细胞中的表达

目的:构建ERK-2基因重组腺病毒载体,检测构建的腺病毒感染原代大鼠生长板软骨细胞的效率以及目的基因的表达。方法:将ERK-2 cDNA亚克隆到腺病毒穿梭载体pAdTrack-CMV中,线性化后与腺病毒骨架质粒pAdEasy-1共同转染E.Col.i B J5183,将筛选、鉴定的重组腺病毒质粒线性化后转染HEK293细胞进行病毒颗粒的包装;流式细胞术检测不同感染复数(MO I)ERK-2重组腺病毒感染原代培养的大鼠肋生长板软骨细胞的效率,W estern b lot检测腺病毒感染的生长板软骨细胞中ERK-2蛋白的表达。结果:成功构建ERK-2重组腺病毒,MO I 50的腺病毒感染原代生长板软骨细胞的效率大于90%,感染的生长板软骨细胞中ERK-2表达显著增加。结论:构建的重组腺病毒可介导ERK-2基因在原代大鼠生长板软骨细胞中高表达。     

蛋白激酶D1对心肌梗死大鼠心肌组织eNOS及Ang1表达的调控作用

目的:探讨蛋白激酶D1(PKD1)对大鼠心肌梗死后心肌组织内皮型一氧化氮合酶(e NOS)及促血管生成素1(Ang1)表达调控的影响.方法:采用冠状动脉左前降支结扎造模方法复制大鼠心肌梗死模型,术后存活达2d以上的心肌梗死大鼠随机被分为模型组、PKD1处理组与CID755673处理组,每组8只.应用逆转录聚合酶链式反应(RT-PCR)分析PKD1及其阻断剂CID755673对大鼠心肌组织中e NOS及Ang1 mRNA表达的影响,应用免疫组化法和免疫印迹法分析e NOS及Ang1的蛋白表达.结果:和心肌梗死模型组相比较,PKD1处理组大鼠心肌组织中e NOS及Ang1的mRNA和蛋白表达水平显著升高(P0.01);和PKD1处理组比较,CID755673处理组大鼠心肌组织中e NOS及Ang1的mRNA和蛋白表达水平显著下降(P0.01),接近于模型组水平.结论:PKD1显著上调大鼠心肌梗死后受损心肌组织中e NOS及Ang1的表达.     

靶向hTERT的shRNA腺病毒载体的构建及其对MCF-7细胞hTERT表达的影响

设计针对人端粒酶逆转录酶催化亚基(hTERT)的干扰靶序列GGAACACCAAGAAGTTCATCT,构建siRNA表达质粒PgenesilhTERT;随后将shRNA表达框克隆到入门载体pENTRTM1A,构建重组质粒pENTR/U6-hTERT-polyA;使用同源重组方式在体外将该表达框重组到腺病毒载体pAd/PL-DEST,得到重组腺病毒质粒pAd/U6 –TERT-polyA;线性化的重组腺病毒质粒在HEK 293细胞内包装为具有感染能力的病毒颗粒rAd-hTERT.同时构建不针对任何基因的shRNA阴性对照rAd-HK,空病毒对照rAd-blank和只含EGFP的rAd -EGFP.四种病毒经酶切、电泳分析表明插入序列正确,腺病毒载体构建成功.Western blotting测定转染后各组hTERT蛋白的表达情况,证实转染rAd-hTERT 48 h后,hTERT的表达明显被抑制.实验表明基于Gateway技术的腺病毒介导的shRNA载体构建成功,rAd-hTERT可有效抑制乳腺癌MCF-7细胞hTERT基因的表达.本研究为针对端粒酶的基因治疗奠定了实验基础.     

腺病毒载体介导GFP基因在鸭胚和雏鸭体内的表达

本试验旨在探讨腺病毒在鸭胚和雏鸭体内的感染情况,为腺病毒作为外源基因载体在活体上进行RNA干涉奠定基础．将GFP基因重组腺病毒（Adv-GFP）静脉注射15日龄鸭胚和10日龄雏鸭,在感染后不N时间采集组织器官,制作冰冻切片,荧光显微镜观察GFP基因在鸭胚和雏鸭组织器官中的表达,结果发现,GFP基因在Adv-GFP感染后24-72h能在鸭胚肝脏及心肌细胞中表达,在感染后1-5d,GFP基因可在雏鸭肝脏、脾脏、腔上囊中稳定表达,本试验首次证明腺病毒作为安全的外源载体,可介导外源基因在鸭胚和雏鸭体内的靶器官中持续稳定表达,为在鸭上进行病毒病的基因治疗提供了基础数据．     

腺病毒载体介导GFP基因在鸭胚和雏鸭体内的表达

本试验旨在探讨腺病毒在鸭胚和雏鸭体内的感染情况,为腺病毒作为外源基因载体在活体上进行RNA干涉奠定基础．将GFP基因重组腺病毒（Adv-GFP）静脉注射15日龄鸭胚和10日龄雏鸭,在感染后不同时间采集组织器官,制作冰冻切片,荧光显微镜观察GFP基因在鸭胚和雏鸭组织器官中的表达．结果发现,GFP基因在Adv．GFP感染后24-72h能在鸭胚肝脏及心肌细胞中表达,在感染后1-5d,GFP基因可在雏鸭肝脏、脾脏、腔上囊中稳定表达．本试验首次证明腺病毒作为安全的外源载体,可介导外源基因在鸭胚和雏鸭体内的靶器官中持续稳定表达,为在鸭上进行病毒病的基因治疗提供了基础数据．     

黄芪丹参配伍提取物经VEGF、Ang1/Tie2通路对心肌梗死大鼠血管新生的病理影响

为观察黄芪、丹参配伍提取物对心肌梗死大鼠VEGF(vascular endothelial growth factor)、血管生成素Ang1及其受体酪氨酸激酶Tie2信号通路的表达影响,对心肌梗死大鼠模型复制成功后分为假手术对照组、模型组、黄芪、丹参配伍提取物低、中、高剂量组,分别予以对应药物灌胃4周。观察大鼠一般生活状态,并分别采用HE染色和免疫组织化学染色测试大鼠心肌组织病理形态结构和VEGF、Ang1、Tie2的蛋白表达。结果表明:模型组心肌结构紊乱伴有炎性侵润,VEGF、Ang1和Tie2蛋白表达较假手术对照组有所增加(P 0. 05);低、中、高剂量组的这些表达随着药物浓度梯度增加,心肌结构逐渐规整,内皮细胞完整、新生血管增多,与模型组比较差异显著(P 0. 05或P 0. 01)。可见黄芪、丹参配伍提取物可以上调心肌梗死大鼠VEGF、Ang1和Tie2的表达,促进血管新生。     

携带p53基因重组腺病毒载体的构建

将pCMVp53重组转移载体经BamHI和NheI酶切,得到p53基因cDNA,然后将cDNA片段克隆到转移载体pCMV5GFP,使其受CMV5启动子的调控,获得pCMV5p53重组转移载体.用该线状重组转移载体与腺病毒右臂DNA经磷酸钙共转染293细胞获得重组腺病毒,经酶联免疫吸附法(ELISA)测定p53蛋白含量,证明外源p53基因在含重组腺病毒的293细胞中得到表达.     

运动对SHR大鼠心肌AngⅡ及AT1 mRNA表达的影响

观察运动对高血压大鼠心肌AngⅡ及AT1mRNA表达的影响,探讨运动对高血压大鼠心肌肥厚作用及机制。雄性SHR大鼠16只,Wistar大鼠8只（C组）,SHR大鼠随机分为安静对照组（S组）和运动组（T组）,每组各8只。T组每日进行90min的无负重游泳,每周6次,共9周。心肌AT1mRNA、AngⅡ、心系数等生化指标来研究和评价。结果显示：1．与C组比较,S组心系数显著升高（P〈0．01）;与S组相比,T组心系数明显降低（P〈0．05）。2．与C组相比,S组心肌和血浆AngⅡ显著升高（P〈0．05,P〈0．01）,心肌和血清NO含量显著降低（P〈0．05,P〈0．01）;与S组相比,T组心肌AngⅡ显著降低（P〈0．01）,心肌和血清NO含量明显升高（P〈0．05）。3．与C组相比,T组和S组心肌AT1mRNA表达显著降低（P〈0．01）;与S组相比,T组心肌AT1mRNA表达偏高,但无统计学意义。得出：长期规律的适宜运动可以明显降低SHR大鼠心系数和心肌AngⅡ含量,提示长期规律的运动能有效改善AngⅡ介导的心肌肥大。     

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.     

Characterization of Ordovician carbonate reservoirs, southeastern Saskatchewan, Canada

The discovery of the prolific Ordovician Red River reservoirs in 1995 in southeastern Saskatchewan was the catalyst for extensive exploration activity which resulted in the discovery of more than 15 new Red River pools. The best yields of Red River production to date have been from dolomite reservoirs. Understanding the processes of dolomitization is, therefore, crucial for the prediction of the connectivity, spatial distribution and heterogeneity of dolomite reservoirs.The Red River reservoirs in the Midale area consist of 3～4 thin dolomitized zones, with a total thickness of about 20 m, which occur at the top of the Yeoman Formation. Two types of replacement dolomite were recognized in the Red River reservoir: dolomitized burrow infills and dolomitized host matrix. The spatial distribution of dolomite suggests that burrowing organisms played an important role in facilitating the fluid flow in the backfilled sediments. This resulted in penecontemporaneous dolomitization of burrow infills by normal seawater. The dolomite in the host matrix is interpreted as having occurred at shallow burial by evaporitic seawater during precipitation of Lake Almar anhydrite that immediately overlies the Yeoman Formation. However, the low δ18O values of dolomited burrow infills (-5.9‰～ -7.8‰, PDB) and matrix dolomites (-6.6‰～ -8.1‰, avg. -7.4‰ PDB) compared to the estimated values for the late Ordovician marine dolomite could be attributed to modification and alteration of dolomite at higher temperatures during deeper burial, which could also be responsible for its 87Sr/86Sr ratios (0.7084～0.7088) that are higher than suggested for the late Ordovician seawaters (0.7078～0.7080). The trace amounts of saddle dolomite cement in the Red River carbonates are probably related to "cannibalization" of earlier replacement dolomite during the chemical compaction.     

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.     

A computer generator for randomly layered structures

AcomputergeneratorforrandomlylayeredstructuresYUJia shun1,2,HEZhen hua2(1.TheInstituteofGeologicalandNuclearSciences,NewZealand;2.StateKeyLaboratoryofOilandGasReservoirGeologyandExploitation,ChengduUniversityofTechnology,China)Abstract:Analgorithmisintrod…     

An Index mechanism for multi-scale view in spatial databases

There are numerous geometric objects stored in the spatial databases. An importance function in a spatial database is that users can browse the geometric objects as a map efficiently. Thus the spatial database should display the geometric objects users concern about swiftly onto the display window. This process includes two operations:retrieve data from database and then draw them onto screen. Accordingly, to improve the efficiency, we should try to reduce time of both retrieving object and displaying them. The former can be achieved with the aid of spatial index such as R-tree, the latter require to simplify the objects. Simplification means that objects are shown with sufficient but not with unnecessary detail which depend on the scale of browse. So the major problem is how to retrieve data at different detail level efficiently. This paper introduces the implementation of a multi-scale index in the spatial database SISP (Spatial Information Shared Platform) which is generalized from R-tree. The difference between the generalization and the R-tree lies on two facets: One is that every node and geometric object in the generalization is assigned with a importance value which denote the importance of them, and every vertex in the objects are assigned with a importance value,too. The importance value can be use to decide which data should be retrieve from disk in a query. The other difference is that geometric objects in the generalization are divided into one or more sub-blocks, and vertexes are total ordered by their importance value. With the help of the generalized R-tree, one can easily retrieve data at different detail levels.Some experiments are performed on real-life data to evaluate the performance of solutions that separately use normal spatial index and multi-scale spatial index. The results show that the solution using multi-scale index in SISP is satisfying.     

Jane Eyre——A Feminist In The 19th Century

In the 19th century the society was controlled by men, and women were just appendants of them, they had not any rights and freedom. But Jane was an exception, she showed some characteristics of early feminist. Jane showed her characteristics of feminism in three aspects: rebellion, equality, and independence. These characteristics were helpful to her success, and feminism is the only way out for women of that time.     

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-