Exendin 4高活性短肽模拟物的筛选及生物活性#br#

以细胞表面胰高血糖素样肽(GLP-1)受体为靶标, 利用噬菌体筛选技术, 通过Exendin-4竞争性洗脱筛选GLP-1受体激动剂, 得到了12肽模拟物EPA. 细胞增殖实验结果表明： EPA和Exendin-4均可促进胰岛β细胞增殖活性, 在40~200 μmol/L内, EPA比Exendin-4的活性高5%~25%; 在100 mmol/L高浓度葡萄糖毒性下, EPA通过抑制bax基因和上调bcl 2基因表达及抑制Caspase 3活性来抑制胰岛β细胞的凋亡; EPA是具有高活性的Exendin-4短肽模拟物, 可通过bcl-2/bax…Caspase 3信号通路抑制线粒体的凋亡.     

Exendin-4高活性短肽模拟物的筛选及生物活性

以细胞表面胰高血糖素样肽(GLP-1)受体为靶标,利用噬菌体筛选技术,通过Exendin-4竞争性洗脱筛选GLP-1受体激动剂,得到了12肽模拟物EPA.细胞增殖实验结果表明:EPA和Exendin-4均可促进胰岛β细胞增殖活性,在40～200μmol/L内,EPA比Exendin-4的活性高5%～25%;在100mmol/L高浓度葡萄糖毒性下,EPA通过抑制bax基因和上调bcl-2基因表达及抑制Caspase-3活性来抑制胰岛β细胞的凋亡;EPA是具有高活性的Exendin-4短肽模拟物,可通过bcl-2/bax…Caspase-3信号通路抑制线粒体的凋亡.     

中草药中胰高血糖素受体拮抗剂的高通量细胞筛选

为筛选胰高血糖素受体拮抗剂，建立了一种高通量筛选胰高血糖素受体拮抗剂的细胞模型.利用PCR方法扩增得到人的胰高血糖素受体 cDNA，并连接到哺乳动物细胞表达载体pCDNA3.1上，构成质粒h glucagons-R/pCDNA3.1，将这种质粒与报告基因质粒(3×CRE/3×MRE/SRE-LUC)共转染到HEK293细胞，通过G418筛选，建立稳定的人胰高血糖素受体拮抗剂筛选细胞株.利用胰高血糖素受体内源激动剂探索和优化了筛选条件，筛选方法合理， Z′因子大于0.6，系统稳定，符合胰高血糖素受体拮抗剂的高通量筛选的要求.     

基于S2细胞建立钙库调控钙离子通道特异分子的筛选模型

应用Flexstation胞质溶胶钙离子检测技术,研究利用果蝇S2细胞建立钙库调控钙离子通道SOC的特异分子的筛选模型.通过三种已知钙通道拮抗剂的检测,发现培养温度为24℃,细胞密度为2×106个/mL时,利用终浓度为2μmol/L的SOC通道激动剂TG可建立稳定的细胞筛选模型.     

胰高血糖素样肽-1受体激动剂功能性报告基因分析系统的构建

用大鼠胰高血糖素样肽-1受体(GLP-1R)基因转染带有增强型绿色荧光蛋白(EGFP)报告基因的CH0细胞,成功地构建了基于G蛋白偶联受体信号传导途径的CHO/EGFP/GLP-1R检测细胞系.该细胞系能功能性表达GLP-1R并且能通过cAMP偶联的EGFP报告基因的表达评价GLP-1R激动剂的活性.利用该细胞系本文进一步对5种GLP-1与金黄色葡萄球菌蛋白A(SPA)不同形式连接的融合蛋白进行了体外活性评价.结果表明,5种融合蛋白的活性均低于天然的GLP-1,将GLP-1融合在SPA的N端与C端融合相比,能显著的提高其生物活性.在融合蛋白之问加入不同长度的重复序列(GGGGS),有助于提高其活性,但间隔序列的长短对活性没有显著的影响.以上结果表明,利用CHO/EGFP/GLP-1R细胞系,能够对GLP-1及其类似物的体外活性进行定性和定量的分析,为筛选和开发长效GLP-1类似物奠定了方法学基础.     

基于细胞的β2肾上腺素能受体激动剂功能性筛选体系在中草药中的应用

为了建立基于细胞的β2肾上腺素能受体激动剂筛选体系,将大鼠β2受体基因克隆人pcDNA3.1载体.转染具有cAMP应答元件调控的绿色荧光蛋白报告基因的中国仓鼠卵巢细胞(CHO),从得到的阳性单克隆细胞中筛选得到一个稳定表达β2受体,并且在受到激动剂刺激后发出剂量依赖绿色荧光强度的细胞株.将此筛选体系用于从中药中筛选具有β2受体激动剂活性的化合物.从几种芸香科药材中得到了辛弗林(synephine),从细辛和附子中得到了去甲乌药碱(higenamine),首次从受体水平证明了它们的β2激动剂活性.此筛选体系适合从大量天然化合物中快速筛选具有β2激动剂活性的成分.     

蛋白酪氨酸磷酸酶SHP2抑制剂高通量筛选模型的建立和应用

 为了建立体外蛋白酪氨酸磷酸酶SHP2抑制剂的高通量筛选模型,筛选潜在的SHP2抑制剂,通过应用大肠杆菌系统克隆表达GST-SHP2 融合蛋白,经过GST-Sepharose 亲和层析分离得到纯化的GST-SHP2 蛋白,建立384 孔板的高通量筛选模型,对48000个有明确结构的小分子化合物进行体外筛选,筛选出75个活性化合物对SHP2的抑制作用大于50%,确定3个化合物具有较高的抑制活性。该筛选模型灵敏、稳定,对SHP2抑制剂药物研发打下了基础。     

蛋白酪氨酸磷酸酶SHP2抑制剂高通量筛选模型的建立和应用

为了建立体外蛋白酪氨酸磷酸酶SHP2抑制剂的高通量筛选模型,筛选潜在的SHP2抑制剂,通过应用大肠杆菌系统克隆表达GST-SHP2融合蛋白,经过GST-Sepharose亲和层析分离得到纯化的GST-SHP2蛋白,建立384孔板的高通量筛选模型,对48000个有明确结构的小分子化合物进行体外筛选,筛选出75个活性化合物对SHP2的抑制作用大于50%,确定3个化合物具有较高的抑制活性。该筛选模型灵敏、稳定,对SHP2抑制剂药物研发打下了基础。     

GLP-1-Fc融合蛋白在DG44细胞中的表达及其质量分析

构建GLP-1-Fc蛋白真核表达载体,并在DG44细胞株中实现表达。利用基因工程等方法构建表达GLP-1-Fc融合蛋白的重组DG44细胞株,通过甲氨喋呤的加压筛选及有限稀释法挑选出表达量较高的单克隆细胞株,经无血清驯化培养获得悬浮生长的细胞株。收集发酵培养上清,通过离心、过滤及亲和层析等方法对目的蛋白GLP-1-Fc进行分离纯化,并利用Dot blot、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、高效液相色谱法、Edman降解等方法进行目的蛋白的质量分析,以建立GLP-1-Fc融合蛋白发酵、纯化工艺,探索该融合蛋白的质量检测方法。本实验成功构建了表达GLP-1-Fc融合蛋白的重组DG44悬浮型细胞株,通过该工艺流程,GLP-1-Fc融合蛋白表达量达到400 mg/L,纯度达95%,分子量约为3.0×10~4。该生产工艺能够获得与理论目标蛋白相一致的、且结构稳定GLP-1-Fc融合蛋白,其质量检测方法稳定可靠。     

黑色素瘤模型的建立与评价

为了快速有效地进行在体药物筛选,研究了以小鼠黑色素实体瘤组织中的原代细胞构建黑色素瘤模型的方法。从荷黑色素瘤小鼠的组织中提取原代肿瘤细胞,与体外培养的B16细胞通过皮下注射分别植入两组C57BL/6小鼠体内,考察两组小鼠肿瘤显现时间和生长速度。结果表明：当接种浓度为5.0×10⁶个/ mL、每只0.2 mL时,小鼠黑色素瘤原代细胞和体外培养的B16细胞成瘤率分别为100%和80%,且小鼠右前肢肿瘤(大小约4 mm³）出现时间分别约在第3天和第8天。紫杉醇对两种模型的初步评价显示,与体外培养的B16细胞模型相比,原代B16细胞模型是体内筛选和评价特定化合物抗肿瘤活性的一种可行而有效的方法,成瘤时间较短、成瘤率高,所得实验数据误差也较小。模型的建立为相关药物评价模型的开发研究提供了一条可借鉴的新途径。     

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.     

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     

An affordable accurate evaluation of the binding conformations predicted by molecular docking studies in bio-system

Franck-Condon factors bridge the gap between theoretical modeling and experimental observations for molecular electronic spectroscopy and electron transfer.Under the displaced harmonic oscillator approximation,multidimensional Franck-Condon factors are decomposed into a product of many one-dimensional(1D)Franck-Condon(FC)factors,and each 1D-FC factor is associated with one Huang-Rhys factor that determines the leading contribution of     

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

正~~