盘基网柄菌发育中的细胞粘附分子及其信号转导

在盘基网柄菌发育早期,DdCAD-1和csA调节了变形虫细胞间的粘着,调控该过程的机制类似于胚胎发育中上皮细胞层的闭合。完成网柄菌发育的一个必需分子是gpl50异嗜性粘附分子。盘基网柄菌β-连环蛋白同源物Aardvark(Aar)的缺乏使细胞间失去粘着连接,Aar也有信号转导功能,调控了前孢子细胞基因的表达。因此,细胞间的粘着是盘基网柄菌发育的一个重要组成部分,并与调控形态发生过程的信号转导有密切相互作用关系。     

细胞色素c在盘基网柄菌细胞凋亡中的作用

细胞色素c在细胞凋亡中发挥着重要的作用,其作用机理在高等真核生物及低等真核生物酵母中已经比较清楚,但在盘基网柄菌(Dictyostelium discoideum)中的作用却没有相关报道.所以我们用western blot和实时荧光定量PCR的方法分别测定了盘基网柄菌前柄细胞和前孢子细胞中细胞色素c的含量及表达量的变化...     

盘基网柄菌细胞的粘附分子

盘基网柄菌（Dictyostelium discoideum)依赖4种类型细胞粘附系统的表达使其多细胞发育顺利进行。在发育初期,由钙结合蛋白DdCAD-1调节EDTA／EGTA敏感的粘着位点。在发育的多细胞聚集阶段,出现EDTA抗性的粘着位点,由分子是80kD蛋白（gp80)通过同嗜性粘着的相互作用末调节细胞的粘着,它的细胞结合位点是一个八肽序列。由分子量150kD蛋白（gp150)通过异嗜性粘着的相互作用来调节多细胞后聚集的细胞粘着。本文详细讨论了gp80和gp150调节细胞粘着的机制。     

gp150蛋白在盘基网柄菌发育中的作用及粘附分子间关系的分析

饥饿的盘基网柄菌进入多细胞发育期 ,在发育早期 ,AK12 7细胞 (gp150突变细胞 )能表达DdCAD 1和 gp80两种粘附分子 ,但它们不足以促进细胞继续发育 ,发育停留在细胞疏松结合阶段。粘附分子 gp150调节的细胞与细胞间的粘着影响了细胞丘“突出”的形成 ,由此影响了盘基网柄菌多细胞发育的形态发生。TL93细胞 (DdCAD 1和gp80突变细胞 )能完成发育。主要原因是在细胞流发育阶段就表达了gp150分子 ,在细胞粘着的功能上有替代DdCAD 1和 gp80的作用。因此 gp150蛋白对盘基网柄菌多细胞发育有着不可或缺的作用     

PKA在盘基网柄菌(Dictyostelium discoideum)多细胞发育中的作用

在盘基网柄菌(Dictyosteliumdiscoideum)多细胞发育中,蛋白激酶A(proteinkinaseA,PKA)发挥多重作用.细胞聚集阶段,PKA调节腺苷酰环化酶的活性,中转cAMP,诱导dut、pdi等一些发育早期的基因表达;参与启动聚集后的细胞分化和形态构成,增强GBF活性,激活前孢子细胞特有基因的表达;它还精密调控前柄细胞特有基因ecmB的表达,准确启动拔顶发育,诱导孢柄和孢子的成熟.子实体形成后,PKA又是维持孢子休眠和保证孢子有效萌发的必需因子.在PKA调控下,盘基网柄菌有条不紊地完成整个发育过程.     

盘基网柄菌中分化诱导因子信号与细胞命运决定

对盘基网柄菌发育过程中分化诱导因子(DIF)的作用及其机制进行了综述,包括DIF对盘基网柄菌前柄细胞、柄细胞分化的作用以及DIF的生物合成、DIF的诱导、降解失活、DIF对细胞命运和细胞比例的调节及其作用机制等。     

盘基网柄菌细胞分化及细胞比例的调控

本文综述了盘基网柄菌(Dictyosteliumdiscoideum)发育过程中调控细胞分化及细胞比例的一些信号分子,包括分化诱导因子(DIF-1、SDF-2)、糖原合成酶激酶(GSK-3)、环状亮氨酸拉链蛋白(rZIP)等,介绍了这些信号分子的功能及其作用机制。     

盘基网柄菌(Dictyostelium discoideum)细胞的分化及其调控

本文综述了盘基网柄菌(Dictyostelium dis-coideum)发育过程中细胞类型的诱导和分化,细胞外cAMP及其四种位于细胞表面的受体及PKA(蛋白激酶A)、GSK-3(糖原合成酶激酶)和STATa等在网柄菌发育过程中的作用。     

盘基网柄菌细胞发育过程中尿囊酸酶的亚细胞定位

利用胶体金免疫电镜技术,观察了盘基网柄菌细胞分化与凋亡过程中胞内尿囊酸酶的位置变化。结果表明,在细胞聚集期细胞产生的尿囊酸酶主要分布于线粒体及周围细胞质内。到了细胞丘时期,尿囊酸酶只特异地存在于发生内自噬的线粒体内,且仅局限于线粒体因内自噬产生的空泡区域,这些发生线粒体内自噬的细胞将分化成前孢子细胞。随着前孢子细胞分化的进行,尿囊酸酶颗粒在细胞内分布逐渐减少,在靠近质膜处的空泡内还能观察到一些酶颗粒;而另一些细胞内,几乎所有的胞器内都能观察到酶颗粒,一直延续至柄细胞形成。从中可以看到尿囊酸酶在将发育成孢子细胞和柄细胞两种类型细胞内的分布位置明显不同,结果提示了尿囊酸酶蛋白与盘基网柄菌细胞分化和凋亡调控途径有密切关系。     

盘基网柄菌DJ-1蛋白的原核表达及抗体制备

盘基网柄菌(Dictyostelium discoideum)是研究神经退行性病变的模式生物,可用以研究帕金森病相关基因DJ-1的作用和致病机理。本研究设计特异性引物扩增了人类DJ-1的盘基网柄菌同源基因片段DAM,并利用酶切位点Bam HⅠ和HindⅢ将DAM插入质粒载体p QE-30产生重组载体pPROF696,序列测定后对DJ-1进行了生物信息学分析。同时,通过电穿孔法将pPROF696转入大肠杆菌M15,并利用IPTG诱导DJ-1蛋白表达。经SDS-PAGE分析、Western-blot印迹和His标签树脂对DJ-1粗蛋白纯化后进行抗体制备,并在大肠杆菌M15转化株和盘基网柄菌转化株与野生型内对抗体进行检验。结果表明:本研究成功构建了盘基网柄菌DJ-1的原核表达载体pPROF696,所制备的盘基网柄菌DJ-1多克隆抗体能成功检测大肠杆菌M15和盘基网柄菌野生型与转化株中的DJ-1蛋白。这为今后检测盘基网柄菌细胞内DJ-1的表达水平,建立其与盘基网柄菌表现型相关性,标记DJ-1的亚细胞定位和进一步研究DJ-1在帕金森病中的作用机理奠定了基础。     

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.     

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.     

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.     

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.