三单位保存的艾氏腹水癌细胞株及克隆细胞蛋白质表达

目的　比较三个单位保存的艾氏腹水癌 (EAC)细胞株及克隆细胞的蛋白质表达。方法　对北京市肿瘤研究所 (北京 )保存的EAC进行克隆培养 ,从中选出 5株克隆 ;对武汉大学保种中心 (武汉 )、山东省医学科学院药物研究所 (山东 )及北京保存的EAC细胞株及 5株克隆细胞的SDS PAGE电泳图谱及免疫组化蛋白质分布进行了对比。结果　武汉、山东及北京保存的EAC的电泳条带数分别为 2 2、2 5及 2 8条 ,而克隆细胞E2G8为 2 6条带。三单位EAC瘤细胞对 5种抗体做免疫组化染色 ,与 1株抗体反应的阳性细胞比例均较高 ,差异无显著性 (P >0 0 5 ) ,与另 4种抗体反应的阳性细胞率差异有显著性 ;5株克隆细胞对 10种抗体作免疫组化染色 ,其中克隆细胞E2G8、E2F4与 7种抗体反应阳性 ,E2C6与 8种抗体反应阳性 ,E1G5、E2B5与 6种抗体反应阳性。克隆细胞一旦与某种抗体反应阳性 ,阳性细胞的比例即在 85 %以上。结论　不同单位保存的EAC细胞株及克隆细胞株蛋白质表达有差异     

一种有效的DNA序列测定方法

比较了单链和双链DNA(ss和dsDNA)模板和两种不同的聚丙烯酰胺凝胶电泳对DNA序列测定的影响。结果表明:在相同的条件下,ssDNA模板明显优于dsDNA模板;缓冲液梯度聚丙烯酰胺凝胶电泳测定DNA序列的长度在40cm长的胶上可达350nt。而一般的线性聚丙烯酰胺凝胶电泳才可测150nt。对于基因组序列分析,采用单链DNA模板和缓冲液梯度聚丙烯酰胺凝胶电泳是一种有效的测定方法。     

枯草芽孢杆菌BF7658噬菌体的研究

从一些工厂分离到6株感染枯草芽孢杆菌BF7658的噬菌体,对它们的形态学和生物学特性以及DNA和结构蛋白进行了比较,电镜观察指出,所有噬菌体的头部外形都呈现椭园形,但其大小和尾部长度有所不同,它们的宿主范围较窄,用限制酶分析,噬菌体DNA分子量在29.9kb和98kb之间,根据解链温度计算出噬菌体DNA的G+C含量在45.1和60.2mol％之间,6株噬菌体的蛋白经SDS-聚丙烯酰胺凝胶电泳测定呈现四条主带和五条次带。     

不同单位保种的S180细胞核型及DNA含量的比较研究

对四个单位保种的S180 细胞经KM 小鼠腹腔传代,体外培养加秋水仙碱,涂片、染色后,显微镜下计数染色体数目:腹水传代的S180 细胞,用70 % 乙醇固定,流式细胞仪测DNA 含量。结果如下:本学部( 本部) ,中国医学科学院药物所( 药物所) ,武汉大学保种中心( 武汉大学) 和北京市肿瘤所( 肿瘤所) 保种的S180 细胞株,其染色体均数分别为62-8 ±22-8 ,69-1 ±21-2,39-9 ±8-26 ,58-7±9-75 条。四单位S180 细胞株染色体数做方差分析表明,除肿瘤所与本部外,其它两单位比较均有显著统计学差异(P< 0-01) 。直方图分析显示主流染色体范围分别为56 ～60 ,61 ～65,41 ～45,61 ～65 条。流式细胞术DNA 含量分析表明肿瘤所S180 DNA含量最多,武汉大学保种的S180 细胞的DNA含量最少。这些结果均证明四个单位的S180 细胞株在一些方面已出现显著差异。     

斑玉蕈酸性磷酸酯酶的酶学性质研究

以斑玉蕈为材料分别从菌盖和菌柄中提取一种酸性磷酸酯酶（ACPase,EC.3.1.3.2）,进一步用硫酸铵沉淀分离,Sephadex G-200柱纯化,从菌盖中分离到3个酶组分,从菌柄中分离到4个酶组分,分别对菌盖和菌柄的酶Ⅰ和酶Ⅰ′进行聚丙烯酰胺凝胶（PAGE）电泳纯度鉴定,均呈现单一酶蛋白带。SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定酶Ⅰ和酶Ⅰ′的相对分子量均为65kDa,SDS-聚丙烯酰胺凝胶电泳及Sephadex G-75凝胶过滤测定分析,酶Ⅰ和酶Ⅰ′均为单亚基蛋白。紫外吸收光谱（UV）测     

商陆蛋白质的纯化及其抗单纯疱疹病毒(Ⅱ型)活性的研究

纯化的商陆蛋白质Ⅰ在SDS-聚丙烯酰胺凝胶电泳中,只显示一条带,与Sigma SDS-6H四种蛋白质标准品迁移率对比,分子量大约为29,000。采用病毒繁殖量抑制测定法(YR测定),观察商陆蛋白质Ⅰ对疱疹病毒Ⅱ型(HSV-2)在Vero细胞中复制的影响,0.15～15μM出现最大抑制率,50％繁殖抑制率为0.32μM。证明商陆蛋白质Ⅰ对HSV-2有明显的抗病毒活性。     

大肠杆菌冷休克蛋白CspC的分离纯化及部分性质测定

经Sepharose Q Fast Flow阴离子交换层析和Superdex 30凝胶过滤层析,从大肠杆菌(Escherichia coli)细胞内分离纯化了一种小分子蛋白质,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)纯度鉴定为单一条带,经质谱分析、N端测序、同源序列比较,确定该蛋白质为大肠杆菌冷休克蛋白CspC.在此基础上,用圆二色光谱测定了其二级结构含量,初步探索了其热稳定性及与单链DNA结合后的构象变化.     

小鼠抗人胰岛素受体单克隆抗体的制备、纯化及鉴定

 M11D杂交瘤细胞株是由人胎盘细胞膜纯化所得胰岛素受体免疫BALB/C小鼠后,取其脾细胞与同系小鼠骨髓瘤细胞株NS-1细胞融合所得。该杂交瘤细胞分泌的抗体经ELISA及放射免疫沉淀法证实为胰岛素受体特异的单克隆抗体。该抗体经Protein A-Sepharose亲和层析分离、纯化,SDS-聚丙烯酰胺梯度凝胶电泳鉴定得分子量分别为53000及23000的两条区带,免疫双扩证明为IgGl。该抗体特异地沉淀125Ⅰ-人胎盘细胞膜胰岛素受体,沉淀经SDS-聚丙烯酰胺凝胶电泳后放射自显影得分子量为135000的特异显影带,与胰岛素受体α亚基分子量相同,说明M11D为抗胰岛素受体α亚基的单克隆抗体。     

人α1/158Ⅴ型(α1c型)干扰素基因在大肠杆菌中的表达及其产物的初步纯化

采用DNA聚合酶链反应(PCR)技术,将本实验室从中国人胎肝细胞染色体DNA中发现和分离的IFN-α1/158V基因的原始克隆,改造成适于进行非融合蛋白原核表达的结构形式,并在大肠杆菌中获得高效表达。测得重组IFN-α1/158V的抗病毒活性为1.9×10~7单位/升菌液。随后又采用以单克隆抗体亲和层析为主的纯化流程对表达产物进行初步纯化,获得了在SDS-聚丙烯酰胺凝胶电泳上呈现单一条带的纯化产物。     

枯草杆菌核糖体基因突变体的翻译产物分析

运用放射性同位素氨基酸,对枯草杆菌核糖体蛋白质基因突变株作体内蛋白质合成脉冲标记,随后用等电点聚焦-SDS聚丙烯酰胺双向凝胶电泳以及荧光自显影等技术,分析和比较了野生型和突变株的翻译产物。发现各菌株的电泳图谱有不同程度的差别,某些蛋白质种类增加,某些种类减少,另外有某些蛋白质的相对浓度有所增减。同是核糖体蛋白质S4突变株,在相同时间脉冲标记的电泳图谱也有不同程度的差别。以上结果反映了核糖体蛋白质基因突变对体内蛋白质合成的影响。     

颌下腺导管细胞与胶原海绵细胞相容性的实验研究

为探讨胶原海绵对颌下腺 (submandibulargland ,SMG)导管细胞的细胞相容性 ,采用HE染色光镜观察及免疫组化观察SMG导管细胞接种于胶原海绵后 ,细胞的生长情况。光镜下可见接种后第 1d细胞数量较少 ,分散于胶原海绵支架中间 ,第 7d细胞数量明显增加 ,免疫组织化学染色抗IV型胶原抗体染色呈阳性 ,说明细胞与支架材料之间已经有细胞外基质产生。胶原海绵具有良好的细胞相容性 ,是一种理想的支架材料。与胶原海绵复合培养 ,颌下腺导管细胞仍可保持良好的增殖能力。     

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed¹ who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate².To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry¹. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments¹.The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection³ in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.     

细胞提取物重编程研究进展

体细胞重编程是在特定的条件下使已分化的细胞转变成为另一种细胞.体细胞重编程的方式主要有体细胞核移植技术、细胞融合技术、细胞提取物处理技术及特定转录因子转染技术.现有研究表明,细胞提取物重编程技术在体细胞重编程中发挥着一定的作用,为此,就该技术的最新研究进展和可能机制作一综述.     

Cell Physician: Reading Cell Motion

Cell motility is an essential phenomenon in almost all living organisms. It is natural to think that behavioral or shape changes of a cell bear information about the underlying mechanisms that generate these changes. Reading cell motion, namely, understanding the underlying biophysical and mechanochemical processes, is of paramount importance. The mathematical model developed in this paper determines some physical features and material properties of the cells locally through analysis of live cell image sequences and uses this information to make further inferences about the molecular structures, dynamics, and processes within the cells, such as the actin network, microdomains, chemotaxis, adhesion, and retrograde flow. The generality of the principals used in formation of the model ensures its wide applicability to different phenomena at various levels. Based on the model outcomes, we hypothesize a novel biological model for collective biomechanical and molecular mechanism of cell motion.     

Tuning Cell Motility via Cell Tension with a Mechanochemical Cell Migration Model

Cell migration is orchestrated by a complicated mechanochemical system. However, few cell migration models take into account the coupling between the biochemical network and mechanical factors. Here, we construct a mechanochemical cell migration model to study the cell tension effect on cell migration. Our model incorporates the interactions between Rac-GTP, Rac-GDP, F-actin, myosin, and cell tension, and it is very convenient in capturing the change of cell shape by taking the phase field approach. This model captures the characteristic features of cell polarization, cell shape change, and cell migration modes. It shows that cell tension inhibits migration ability monotonically when cells are applied with persistent external stimuli. On the other hand, if random internal noise is significant, the regulation of cell tension exerts a nonmonotonic effect on cell migration. Because the increase of cell tension hinders the formation of multiple protrusions, migration ability could be maximized at intermediate cell tension under random internal noise. These model predictions are consistent with our single-cell experiments and other experimental results.     

Cell Cycle Markers for Live Cell Analyses

Many cellular processes are regulated by cell cycle dependent changes in protein dynamics and localization. Studying these changes in vivo requires methods to distinguish the different cell cycle stages. Here we demonstrate the use of DNA Ligase I fused to DsRed1 as an in situ marker to identify S phase and the subsequent transition to G2 in live cells. Using this marker, we observed changes in the nuclear distribution of Dnmt1 during cell cycle progression. Based on the different nuclear distribution of DNA Ligase I and Dnmt1 in G2 and G1, we demonstrate that the combination of both proteins allows the direct discrimination of all cell cycle phases using either immunostainings or fusions with fluorescent proteins. These markers are new tools to directly study cell cycle dependent processes in both, fixed and living cells.