转录因子C／EBPβ的研究进展

C／EBPs增强子结合蛋白是核转录因子,其作用范围广泛,既参与正常的生理代谢过程,又与多种疾病的发生和发展相关,其作用方式多样,对转录有正、负调控作用。C／EBPβ是其第二位成员主要通过对靶细胞基因转录的调节,参与细胞的增殖与分化、肿瘤的发生与凋亡等重要生命活动;其功能受到蛋白酶降解、磷酸化、蛋白质相互作用等多种途径的调控。本文就C／EBPs的调控机理及其与肿瘤的关系综述如下。     

小分子物质对维持胚胎干细胞未分化状态的调控作用

胚胎干细胞作为一种具有多潜能和高度自我更新能力的种子细胞,己被广泛地应用于医学研究领域。在体外培养条件下,胚胎干细胞可被诱导分化为三个胚层来源的组织细胞,故被看作为最具有应用前景的种子细胞。近年来,对于在体外培养条件下如何维持胚胎干细胞的多能性即使其较长时期的处于未分化状态成为研究热点,其中一些天然存在或人工合成的小分子物质可通过作用于某些特定的靶信号通路,调控胚胎干细胞的分化命运。本文概述了几种小分子物质的最新研究进展,并对小分子物质在成体多分化潜能胚胎样干细胞分化调控方面的应用前景进行评述。     

转录因子C/EBPβ的翻译后修饰

CCAAT增强子结合蛋白β(CCAAT enhancer-binding proteinβ,C/EBPβ)是转录因子C/EBP家族的重要成员。它通过对靶基因转录的调节,参与细胞增殖与分化、肿瘤发生与凋亡、细胞周期调控等重要生命活动,同时这些功能受到磷酸化、乙酰化、泛素化等多种翻译后修饰的调控。本文综述近年来有关C/EBPβ的翻译后修饰的研究进展。这些研究不仅为人们认识C/EBPβ的调控机制提供了一个新的角度,同时也可能为肥胖及相关代谢疾病的发生和治疗提供新的理解。     

新疆维吾尔族妇女子宫颈鳞癌组织中C/EBPβ蛋白的表达及意义

目的:研究C/EBPβ蛋白在新疆维吾尔族妇女子宫颈鳞癌组织中的表达及临床意义。方法:采用免疫组织化学S-P法对38例子宫颈鳞癌组织、28例慢性子宫颈炎组织中C/EBPβ蛋白的表达进行检测。结果:新疆维吾尔族妇女子宫颈鳞癌组织与慢性子宫颈炎C/EBPβ蛋白的表达无显著性差异(P>0.05);在不同病理分级的肿瘤组织中C/EBPβ蛋白的表达有显著性差异(P<0.05)。结论:C/EBPβ蛋白表达可能与新疆维吾尔族子宫颈鳞癌不同病理分级相关。     

miR-191通过调控C/EBPβ转录影响猪前体脂肪细胞分化

对实验室前期Solexa结果进行深入挖掘,结合生物信息学分析从不同发育阶段猪皮下脂肪组织差异表达的miRNAs中筛选出高丰度差异极显著的候选miR-191.采用腺病毒过表达miR-191,实时定量PCR、Western blot及双荧光素酶报告基因检测等技术方法,初步研究miR-191对猪前体脂肪细胞分化的影响.结果发现,miR-191随着猪前体脂肪细胞的分化表达量逐渐增加.与对照组相比,过表达miR-191的猪前体脂肪细胞中miR-191转录本显著增加,并引起CCAAT增强子结合蛋白β(C/EBPβ)、PPARγ和aP2的mRNA水平降低,抑制了猪前体脂肪细胞分化.同时,Western blot结果显示,与对照组相比过表达miR-191的猪前体脂肪细胞在48 h C/EBPβ蛋白水平下降了55%.更重要的是,通过TargetScan等算法正向筛选以及MicroInspector反向筛选联合获得miR-191候选靶基因,经双荧光素酶报告基因检测结果证实,miR-191可直接作用于C/EBPβ3′UTR,从而降低萤火虫荧光素酶活性.综上所述,miR-191可能通过抑制脂肪细胞分化早期标志基因C/EBPβ的表达,从而抑制了猪前体脂肪细胞的分化.     

C/EBPα在非小细胞肺癌中的表达及其与微血管密度的关系

目的:探讨C/EBPα在非小细胞肺癌(non-small cell lung cancer,NSCLC)组织中的表达及其与肿瘤微血管密度(microvessel density,MVD)的关系.方法:应用免疫组织化学EnVision法检测40例手术切除的NSCLC组织及其配对的40例距肿瘤＞5 cm以上癌旁正常肺组织中C/EBPα蛋白的表达,并分析C/EBPα在NSCLC中的表达与其临床病理特征的关系.采用CD34标记肺癌组织中的肿瘤微血管,分析C/EBPα的表达与肿瘤MVD的关系.结果:NSCLC组织中C/EBPα的阳性表达率明显低于癌旁正常肺组织(P＜0.05).C/EBPα与NSCLC患者的年龄、性别、TNM分期及有无淋巴结转移均无关(P＞0.05),与NSCLC组织的分化程度及病理类型有关(P＜0.05).在NSCLC组织中,C/EBPα蛋白表达阳性组MVD明显低于C/EBPα蛋白表达阴性组(P＜0.05).结论:C/EBPα在NSCLC中的低表达可能通过调节MVD介导NSCLC的发生和发展.     

原代培养大鼠脂肪前体细胞分化过程中PPARγ和C/EBPαmRNA的表达(简报)

脂肪前体细胞是一类具有增殖分化能力的单能干细胞,在体内多种因素的影响下,脂肪前体细胞聚脂分化为成熟脂肪细胞。研究表明,脂肪前体细胞的聚脂分化过程受到一系列基因的调控,其中.过氧化物酶体增殖体激活受体(peroxisome prolifera- tors-activated receptor gamma,PPARγ)与CCAAT增强子结合蛋白α(CCAAT/enhancer binding protein al-     

核转录因子C/EBPβ的研究进展

C／EBPβ是转录因子C／EBPs(CCAAT enhancer binding proteins)家族的重要成员,其C端具有高度保守的DNA结合域和二聚化功能域。它主要通过对靶细胞基因转录的调节,参与细胞增殖与分化、肿瘤发生与凋亡、机体炎症反应等重要生命活动;其功能受到蛋白酶降解、磷酸化、蛋白质相互作用等多种途径的调控。本综述有关C／EBPβ的生物学功能及其调控机理近年来的一些研究进展。     

维持胚胎干细胞不分化状态的分子机制

胚胎干细胞（embryonic stem cell,ESC）是指从早期胚胎的囊胚内细胞团（inner cell mass,ICM）分离出来的具有自我更新和多向分化潜能的细胞,目前被广泛地应用于基础研究和临床应用研究等生命科学领域。ESC在体外培养过程中维持不分化状态是其应用的前提与基础,阐明这个分子机制非常必要。文章总结了维持hESC未分化状态机制的最新进展,主要介绍在维持ESC不分化过程中,分化抑制因子LIF、Oct-3/4及Nanog等的重要作用。     

低氧抑制C2C12细胞的体外分化

目的:研究低氧环境对C2C12细胞分化的影响,为探讨肌肉的发生和骨骼肌的损伤修复机理提供理论依据.方法:培养C2C12细胞,分别在常氧和低氧(3%O2)条件下诱导分化.免疫细胞化学方法检测成肌细胞终末分化的标志蛋白MI-IC(肌球蛋白重链)的表达;Western blot检测MHC以及MRFs(成肌调控因子)的表达.结果:在常氧条件下诱导分化的C2C12细胞融合形成肌管并表达MHC蛋白,而在低氧条件下培养的C2C12细胞几乎很少融合形成肌管并表达MHC蛋白;同时低氧下调了C2C12细胞中MRFs的表达.结论:低氧抑制了C2C12细胞的体外分化.     

分离MEF和ES细胞的差速贴壁法研究(简报)

胚胎干细胞的分化控制是胚胎干细胞研究的一个重要方面。由于常规的拟胚体诱导途径是在形成拟胚体后才开始进行诱导分化,受多种胚层的共同作用使得我们无法简便探索诱导分化的机制。而且用这种方法进行的诱导分化试验的结果检测比     

小鼠胚胎干细胞分化为血管内皮细胞的永生化研究

本文探讨了小鼠胚胎干细胞(ES细胞)诱导分化的血管内皮细胞永生化。在体外培养系统中,以维甲酸(RA)和转化生长因子-β1(TGF-β1)诱导小鼠胚胎干细胞(ES细胞)的拟胚体(EB)分化为“圆形细胞”和由这些“圆形细胞”组成的血管样结构。经光学和扫描电镜及免疫荧光等法分析检测,证明组成血管样结构的细胞具有专一性vWF荧光染色,表明是血管内皮样细胞。利用脂质体将人端粒酶催化亚基逆转录酶(hTERT)基因转染诱导分化中的“圆形细胞”。应用Dot-blot,RT-PCR,Western blot及免疫组织化学等方法分析、观察和证明了诱导分化的组成血管样结构的园形细胞和被hTERT基因转染的“圆形”细胞的形态和生物学特性。结果表明,携带hTERT基因的从ES细胞分化来的圆形细胞在体外可大量增殖,持续传代,95%具有血管内皮细胞的一些特有标志和管道化生长特性。因此,通过人端粒酶基因的转染途径可解决由ES细胞诱导分化而来的内皮细胞扩增和永生化问题,为构建组织工程化血管及其它人工血管的内皮化提供种子细胞来源打下基础。     

HDC细胞与MESPU-13细胞形成嵌合鼠能力的比较研究

ES细胞嵌合能力的强弱是人们利用ES细胞获得转基因小鼠时十分关心的问题。本文通过囊胚显微注射法将15个左右ES细胞注入C57 BL/6 J品系小鼠3.5天囊胚的囊胚腔中观察嵌合鼠毛色嵌合情况,统计嵌合鼠的出生率;以及用葡萄糖磷酸异构酶(GPI)电泳法检测ES细胞在嵌含鼠体内各种组织和器官的嵌合情况,对于HPRT缺陷(HDC)细胞和MES-PU-13细胞的嵌合能力我们作了较详细的研究,结果表明MESPU-13细胞嵌合能力较强,而HDC细胞嵌合能力较弱,并讨论分析了这种结果的原因。     

Expression and function of CCAAT/enhancer binding proteinbeta (C/EBPbeta) LAP and LIP isoforms in mouse mammary gland, tumors and cultured mammary epithelial cells

CCAAT/Enhancer binding proteins (C/EBPs) play important roles in the regulation of cell growth and differentiation. This study investigated the expression and function of C/EBPbeta isoforms in the mouse mammary gland, mammary tumors, and a nontransformed mouse mammary epithelial cell line (HC11). C/EBPbeta mRNA levels are 2-5-fold higher in mouse mammary tumors derived from MMTV/c-neu transgenic mice compared with lactating and involuting mouse mammary gland. The "full-length" 38 kd C/EBPbeta LAP ("Liver-enriched Activator Protein") isoform is the predominant C/EBPbeta protein isoform in mammary tumor whole cell lysates, however, the truncated 20 kd C/EBPbeta LIP ("Liver-enriched Inhibitory Protein") isoform is also present at detectable levels (mean LAP:LIP ratio 5.3:1). The mammary tumor C/EBPbeta LAP:LIP ratio decreases 70% (from 5.3:1 to 1.6:1) when lysate preparation is switched from a rapid whole cell lysis protocol to a multistep nuclear/cytoplasmic fractionation protocol. In contrast to mammary tumors, only the C/EBPbeta LAP isoform is detectable in the mammary gland whole cell and nuclear lysates; the truncated "LIP" isoform is undetectable regardless of isolation protocol. Ectopic over expression of C/EBPbeta LIP or C/EBPbeta LAP did not alter HC11 growth rates. However, C/EBPbeta LIP over expressing HC11 cells (LAP:LIP ratio of approximately 1:1) exhibited a consistent 2-4 h delay in G(0)/S phase transition. C/EBPbeta LIP overexpressing HC11 cells did not express beta-casein mRNA (mammary epithelial cell differentiation marker) in response to lactogenic hormones. This defect in beta-casein expression was not corrected by carrying out the differentiation protocol in the presence of an artificial extracellular matrix. These results demonstrate that the "full-length" C/EBPbeta LAP isoform is the predominant C/EBPbeta protein isoform expressed in mouse mammary gland in vivo and mouse mammary epithelial cell cultures in vitro. C/EBPbeta LIP detected in mammary tumor lysates may result from in vivo production or ex vivo isolation-induced proteolysis of C/EBPbeta LAP. Ectopic overexpression of C/EBPbeta LIP (LAP:LIP ratio of approximately 1:1) inhibits mammary epithelial cell differentiation (beta-casein expression).     

合成洗涤剂对人和哺乳动物细胞的诱变性研究

各种合成洗涤剂(洗衣粉,洗发膏,餐具洗涤剂等)产量日增。在日常生活中使用越来越普遍。洗涤剂直接或间接通过环境污染对人类健康产生影响,特别是潜在的致突变性引起公众的普遍注意。而现有的研究结果并不一致。本研究选用三种型号的合成洗涤剂,以小鼠生殖细胞染色体畸变和骨髓细胞微核率及离体的人类细胞和中国仓鼠细胞的染色体畸变和姐妹染色单体交换(SCE)为测定指标,系统地对合成洗涤剂的诱变活     

C/EBPalpha S248A mutation reduces granulocytic differentiation in human leukemic K562 cells

Phosphorylation of C/EBPα can either lead to granulocytic differentiation or a block in granulopoiesis. This dichotomy in effect is dependent on the upstream signaling pathway and the phosphorylation site in C/EBPα. Ras signaling induced phosphorylation of S248 residue of C/EBPα is known to enhance granulocytic differentiation. In this study, using β-estradiol inducible stable cell lines we show that the point mutation of phosphorylation site S248 in C/EBP disrupts the CD11b and GCSFr expression and subsequently reduces the differentiation of leukemic K562 cells. Based on our observations in the present study, we conclude that S248A mutation of C/EBPα leads to a reduction of granulocytic differentiation markers and a block in differentiation at the morphological level.     

A modified chromatin-immunoprecipitation protocol for silkmoth ovarian follicular cells reveals C/EBP and GATA binding modes on an early chorion gene promoter

Standard Chromatin immunoprecipitation protocols have been designed to suit studies performed on cell line cultures or yeast cells growing in liquid cultures. In these cases cross-linking/fixation takes place directly in the growing medium of the cells by the addition of a general fixation reagent. When applied on whole isolated silkmoth follicles, this procedure results in poor release of follicular cells from the basal membrane and lower yield of cross-linked chromatin. We present a modification to the standard protocol, where detachment of follicular cells from the basal membrane of the egg and nuclei isolation precedes formaldehyde-mediated cross-linking. We also discuss application of the modified method for the identification of distinct BmC/EBP and BmGATAβ binding modes on a chorion gene promoter from the Er1.A/B early gene pair.     

Utility of a C57BL/6 ES line versus 129 ES lines for Targeted Mutations in Mice

Inbred ES lines, though useful for generating targeted mutations in mice, are used infrequently. To appreciate the relative efficiency of inbred ES lines, a C57BL/6 ES line was compared with 129 strain ES lines for effectiveness in chimera formation leading to the establishment of targeted mutations in mice. Data from a transgenic facility spanning 7 years were collected. C57BL/6 ES cells injected into Balb/c embryos results in lower coat color chimerism than do 129 ES cells injected into C57BL/6 embryos. Combined data indicate that five independent targeted C57BL/6 clones should be injected as compared to three independent 129 clones to generate enough chimeras to effectively test for germ-line transmission. Thus, although less efficient than 129 ES lines, the C57BL/6 ES line is a relatively competent line and useful for the routine generation of targeted mutations in mice on a defined genetic background.     

Sequences near both termini of the C/EBPβ mRNA 3＇ untranslated region are important for its tumor suppression activity

The 3＇ untranslated region （3＇ UTR） of eukaryotic mRNA is an important regulation element that affects not only mRNA translation, but also cell growth. We had found that the 3＇ UTR of CCAAT-enhancerbinding protein β （C/EBPβ） mRNA had tumor suppression activity. Herein, we reported that deletion of two short sequences at both termini of the C/EBPβ 3t UTR reduced the tumor suppression activity of this 3＇ UTR, as demonstrated by reduced cell growth, colony formation ability, and tumorigenicity in nude mice. It is noteworthy that the only deletion of a single such sequence was enough for the reduction of tumor sup- pression effect, and the reducing effect of deletion of the sequence near 3r terminus was stronger. Therefore, specific short sequences in the C/EBPβ 3＇ UTR are crucial for the tumor suppression activity of C/EBPβ.     

Nucleolar retention of a translational C/EBPα isoform stimulates rDNA transcription and cell size

The messenger RNA of the intronless CEBPA gene is translated into distinct protein isoforms through the usage of consecutive translation initiation sites. These translational isoforms have distinct functions in the regulation of differentiation and proliferation due to the presence of different N‐terminal sequences. Here, we describe the function of an N‐terminally extended protein isoform of CCAAT enhancer‐binding protein α (C/EBPα) that is translated from an alternative non‐AUG initiation codon. We show that a basic amino‐acid motif within its N‐terminus is required for nucleolar retention and for interaction with nucleophosmin (NPM). In the nucleoli, extended‐C/EBPα occupies the ribosomal DNA (rDNA) promoter and associates with the Pol I‐specific factors u pstream‐b inding f actor 1 (UBF‐1) and SL1 to stimulate rRNA synthesis. Furthermore, during differentiation of HL‐60 cells, endogenous expression of extended‐C/EBPα is lost concomitantly with nucleolar C/EBPα immunostaining probably reflecting the reduced requirement for ribosome biogenesis in differentiated cells. Finally, overexpression of extended‐C/EBPα induces an increase in cell size. Altogether, our results suggest that control of rRNA synthesis is a novel function of C/EBPα adding to its role as key regulator of cell growth and proliferation.