利用流式细胞仪分选拟南芥根尖发育早期非根毛细胞

建立了应用流式细胞仪分选植物特定类型细胞的方法。以拟南芥(Arabidopsis thaliana)Wer::GFP转基因株系为材料,用激光共聚焦显微镜鉴定GFP的表达位置,采用酶解法制备拟南芥根尖原生质体,应用流式细胞仪荧光激活细胞分选技术(FACS)分选收集GFP阳性细胞,并提取细胞的RNA。结果表明,Wer::GFP转基因株系仅在根表皮发育早期的非根毛细胞中表达GFP;利用酶解法制备的根尖原生质体数目较多;从FACS分选收集的细胞中提取的RNA质量较好,可用于研究特定类型细胞的基因表达谱。应用流式细胞仪分选拟南芥非根毛细胞的方法为研究植物特定类型细胞的基因表达谱及基因功能奠定了技术基础。     

利用流式细胞仪分选拟南芥根尖发育早期非根毛细胞

建立了应用流式细胞仪分选植物特定类型细胞的方法。以拟南芥（Arabidopsis thaliana）Wer：：GFP转基因株系为材料,用激光共聚焦显微镜鉴定GFP的表达位置,采用酶解法制备拟南芥根尖原生质体,应用流式细胞仪荧光激活细胞分选技术（FACS）分选收集GFP阳性细胞,并提取细胞的RNA。结果表明,Wer：：GFP转基因株系仅在根表皮发育早期的非根毛细胞中表达GFP;利用酶解法制备的根尖原生质体数目较多;从FACS分选收集的细胞中提取的RNA质量较好,可用于研究特定类型细胞的基因表达谱。应用流式细胞仪分选拟南芥非根毛细胞的方法为研究植物特定类型细胞的基因表达谱及基因功能奠定了技术基础。     

《Molecular Plant》文章中文摘要

拟南芥配子体发育相关的减数分裂和有丝分裂细胞周期突变体（综述）;基于S-核酸酶的自交不亲合的生化模型（综述）;GEX3,在拟南芥雄配子体和卯细胞中表达,对于珠孔花粉管定向生长非常重要同时在胚胎早期发育中起作用;绒毡层降解阻滞对水稻花粉发育过程中的脂肪代谢和基因调控很关键;拟南芥双受精涉及阻断卯细胞的多精受精而非中央细胞……     

山杏叶乙酸乙酯提取物调控乳腺癌MCF7细胞增殖和凋亡的机制研究

为探究山杏叶乙酸乙酯提取物对乳腺癌MCF7细胞株增殖和凋亡的影响,本研究采用CCK8法检测山杏叶提取物对MCF7细胞增殖的抑制作用,流式细胞仪检测细胞凋亡率,倒置荧光显微镜和流式细胞仪分别检测胞内活性氧(ROS)的水平变化,RT-PCR检测细胞周期及凋亡相关基因的表达情况,试剂盒检测caspase-3的活性。实验表明,山杏叶提取物可降低MCF7细胞存活率,促进细胞凋亡,增加胞内ROS水平。同时上调和Bax,下调Bcl-2,增强caspase-3活性,并降低CDK4、CyclinE和CyclinD1的表达。综上说明山杏叶提取物可通过调控周期蛋白的表达来抑制MCF7细胞的增殖,并通过caspase途径和提升ROS水平来诱导MCF7细胞的凋亡。     

细胞周期分子机制的成功探索——2001年诺贝尔生理及医学奖部分工作介绍

细胞周期是指连续分裂的细胞从一次有丝分裂结束到下一次有丝分裂完成所经历的整个序贯过程.在这一过程中,细胞的遗传物质(DNA)经过复制平均分配到两个子细胞中.细胞周期中每一事件都是有规律、精确地发生,并且在时间与空间上受到严格调控.细胞周期中最关键的三类调控因子是：cdc基因、周期蛋白依赖性激酶(CDKs)及细胞周期蛋白(cyclin).这些调控因子的发现对肿瘤学及发育生物学的发展都有重要的理论和实践意义.     

流式细胞仪分选转基因斑马鱼胚胎荧光标记细胞的一种快速方法

荧光蛋白在特异组织和器官表达的转基因斑马鱼已经在发育生物学和疾病模型研究中得到了广泛的应用。这些转基因系有助于追踪和分析数量较少的细胞群。但是如果要分离得到这些细胞来定量分析mRNA或蛋白质的表达情况比较困难。利用流式细胞仪分选这些荧光标记细胞是一种解决办法。此方法在不同的实验室中被广泛地应用。也有相关流程的介绍。但是流程一般较为繁琐,操作比较困难。该文以从转基因斑马鱼Tg（Kdrl：EGFP）中分选绿色荧光蛋白阳性的血管内皮细胞为例,介绍利用流式细胞仪分选转基因斑马鱼荧光标记细胞的实验流程和技术要点。该文作者在前人工作的基础上结合大量的实验经验．发展并优化了一套操作简便、效率较高的流程来分选这些细胞,在此做详细的介绍给大家以供参考。     

利用激光扫描共聚焦显微镜研究拟南芥气孔发生与发育

通过激光扫描共聚焦显微镜,利用不同种类(波长)的激光研究拟南芥叶片气孔发生与发育。结果表明,利用紫外激光(351nm)扫描可以清楚观察到拟南芥表皮各种细胞及其发生发育的形态变化,包括表皮毛细胞、副卫细胞、保卫细胞、铺垫表皮细胞等。气孔发生过程中,首先原表皮细胞不对称分裂产生拟分生组织和副卫细胞,接着分化出保卫细胞母细胞,进一步发育形成保卫细胞,最终形成气孔器。气孔分化完成后,保卫细胞在紫外激光下不产生荧光,但利用蓝光激发(488nm)辅助荧光素染色,可清晰地看到保卫细胞。结果表明,激光扫描共聚焦显微镜在拟南芥叶表皮细胞形态研究上有独特的功能。     

荧光染料Hoechest 33342与干细胞研究

Hoechest 33342是一种可以与DNA结合的荧光染料,在细胞周期研究方面得到广泛的应用。最近研究发现,某些癌细胞和干细胞可以将进入细胞的Hoechest 33342排出细胞外,利用流式细胞仪可以将这些不着色的细胞加以分离。现在已经从许多组织中分离得到了这种细胞。许多研究也提出了利用该方法分离干细胞的可能性。     

核内再复制分子机制研究进展

核内再复制是指细胞没有经历有丝分裂而形成特殊的多倍体核的现象。这是由于细胞周期没有进入M期并多次重复进入S期所致,其主要特征是MPF失活及S期CDKs激酶活性呈周期性振荡。核内再复制现象普遍存在于动物和植物中,在高代谢活性组织的细胞及最终进行高度分化的细胞中最常见。对细胞迅速生长和增殖有着重要的意义。如何阻止细胞有丝分裂的进行,进而引发核内再复制的机制仍在研究中。本文对植物及哺乳动物细胞中核内再复制的产生、调控机制及体外诱导方式等进行了综合评述。     

E1/E3缺失型腺病毒载体引起细胞周期G_2/M阻滞

腺病毒载体广泛应用于基因治疗和转基因研究 ,目前常用的E1 E3缺失型复制缺陷腺病毒载体虽然失去了病毒复制必需的E1基因 ,但载体上的其它病毒基因仍能在宿主细胞内表达 .为研究这些基因对细胞的毒性作用 ,选择了 3种携带没有明显细胞毒性外源基因的腺病毒载体 ,观察感染 2种肿瘤细胞后细胞核形态改变 ,并用流式细胞仪检测细胞周期及凋亡情况 .发现大剂量重组缺陷型腺病毒感染细胞后引起细胞变圆 ,核增大 ,细胞周期阻滞于G2 M期 ,继而染色质凝聚 ,细胞发生坏死或凋亡 ;各种腺病毒载体造成G2 M阻滞所需感染量不同 ,但都随时间延长和感染量增加而加重 .这些结果提示腺病毒基因对细胞的影响是多方面的 ,在以此类病毒载体进行基因转移和基因治疗的研究中 ,精确滴定病毒滴度和转导效率非常重要 ,腺病毒基因表达造成的毒副作用给此类研究增加了变数     

Elongator complex is critical for cell cycle progression and leaf patterning in Arabidopsis

The mitotic cell cycle in higher eukaryotes is of pivotal importance for organ growth and development. Here, we report that Elongator, an evolutionarily conserved histone acetyltransferase complex, acts as an important regulator of mitotic cell cycle to promote leaf patterning in Arabidopsis. Mutations in genes encoding Elongator subunits resulted in aberrant cell cycle progression, and the altered cell division affects leaf polarity formation. The defective cell cycle progression is caused by aberrant DNA replication and increased DNA damage, which activate the DNA replication checkpoint to arrest the cell cycle. Elongator interacts with proliferating cell nuclear antigen (PCNA) and is required for efficient histone 3 (H3) and H4 acetylation coupled with DNA replication. Levels of chromatin-bound H3K56Ac and H4K5Ac known to associate with replicons during DNA replication were reduced in the mutants of both Elongator and chromatin assembly factor 1 (CAF-1), another protein complex that physically interacts with PCNA for DNA replication-coupled chromatin assembly. Disruptions of CAF-1 also led to severe leaf polarity defects, which indicated that Elongator and CAF-1 act, at least partially, in the same pathway to promote cell cycle progression. Collectively, our results demonstrate that Elongator is an important regulator of mitotic cell cycle, and the Elongator pathway plays critical roles in promoting leaf polarity formation.     

Effect of auxin on the mitotic cell cycle in cultured leaf segments at different stages of development in wheat

Young leaves of Triticum timopheevi Zukh. show a defined gradient of development. One-mm-long sections from such leaves were cultured in vitro. At a low concentration of exogenous auxin, cells in the most basal, highly meristematic explants divided readily in culture, but in the absence of auxin they soon ceased dividing and were arrested in G1 and G2 of the mitotic cell cycle. In the region adjoining the meristem, where most cells were arrested in G1, very high concentrations of auxin had to be applied to reinitiate cell division, i.e. stimulate transitions from G1 to S-phase and from G2 to mitosis. Above this potentially auxin-responsive region, which represented less than 50% of the total leaf length, there followed tissue, which, when excised, showed nuclear DNA replication in a number of cells in the absence of auxin. However, the cells did not complete the mitotic cycle, either in the absence or presence of exogenous auxin. We suggest this loss of responsiveness is correlated with an uncoupling of auxin from the control of the cell cycle.     

Cellular responses of leaf explants of Cocos nucifera L. in vitro

Leaf explants of Cocos nucifera L. (coconut palm) were studied in vitro in order to establish whether or not rapid cellular changes contribute to the well known recalcitrance of coconut cells in tissue culture. Segments from the base of immature leaves were cultured on modified Eeuwens' medium at 30°C in darkness. The mitotic index, nuclear DNA amounts, cell and nuclear size were measured both before and during culture (from 0 to 70 days). There was no basipetal gradient of cell division in immature coconut leaves; the mitotic index never exceeded 2% and showed neither a positional nor temporal relationship with leaf development. Moreover the vast majority of cells were in G1 of the cell cycle. This cell cycle pattern was maintained for most of the period in culture although at 70 days there was an increase in the proportion of cells in S- and G2-phases consistent with low rates of callus formation. The nuclear: cell size ratio was constant in cells within the immature leaf irrespective of developmental age. However upon transfer to culture media, cell size but not nuclear size increased. We suggest that this uncoupling of cell and nuclear size disrupts cell co-ordination and is a key contributor to recalcitrant cellular behaviour of this species in vitro.     

Control of the cell cycle by neurotrophins: lessons from the p75 neurotrophin receptor

Although traditionally little attention has been paid to the interplay between neurotrophins and the cell cycle, a number of recent findings suggest an important role for these growth factors in the regulation of this aspect of the cellular physiology. In this article, we review the evidence from a number of studies that neurotrophins can influence cell cycle progression or mitotic cycle arrest both in the nervous system as well as in other cell types. The contrary response of different cells to neurotrophins in terms of cell cycle regulation derives in part from the fact that these factors use two different receptor types to transmit their signals: members of the Trk family and the p75 neurotrophin receptor (p75NTR). With this in mind, we outline the current state of our knowledge regarding the molecular basis underlying the control of cell cycle progression by neurotrophins. We focus our interest on the receptors that transduce these signals and, in particular, the striking finding that p75NTR interacts with proteins that can promote mitotic cycle arrest. Finally, we discuss the mechanisms of cell death mediated by p75NTR in the context of cell cycle regulation.     

Stathmin Prevents the Transition from a Normal to an Endomitotic Cell Cycle during Megakaryocytic Differentiation

Physiological polyploidy is a characteristic of several cell types including themegakaryocytes (MK) that give rise to circulating blood platelets. MK achieve polyploidy byswitching from a normal to an endomitotic cell cycle characterized by the absence of late mitoticstages. During an endomitotic cycle, the cells enter into mitosis and proceed normally throughmetaphase and early anaphase. However, late anaphase, telophase and cytokinesis are aborted. Thisabortive mitosis is associated with atypical multipolar mitotic spindles and limited chromosomesegregation. Stathmin is a microtubule-depolymerizing protein that is important for the regulation ofthe mitotic spindle and interfering with its expression disrupts the normal mitotic spindle and leadsto aberrant mitotic exit. As cells enter mitosis, the microtubule depolymerizing-activity of stathminis switched-off, allowing microtubules to polymerize and assemble into a mitotic spindle.Reactivation of stathmin in the later stages of mitosis is necessary for the disassembly of the mitoticspindle and the exit from mitosis. Previous studies had shown that stathmin expression isdownregulated as MK become polyploid and inhibition of its expression in K562 cells increasestheir propensity to become polyploid. In this report, we describe our studies of the mechanism bywhich stathmin plays its role in MK polyploidization. We show that stathmin overexpressionprevents the transition from a mitotic cycle to an endomitotic cycle as determined by a decrease inthe number of multipolar mitotic spindles. These observations support a model in whichdownregulation of stathmin expression in megakaryocytes and other polyploid cells may be acritically important factor in endomitosis and polyploidy.     

Dual functions of Nicotiana benthamiana Rae1 in interphase and mitosis

Rae1 performs multiple functions in animal systems, acting in interphase as an mRNA export factor and during mitosis as a mitotic checkpoint and spindle assembly regulator. In this study we characterized multiple functions of Rae1 in plants. Virus-induced gene silencing of Nicotiana benthamiana Rae1 , NbRae1 , which encodes a protein with four WD40 repeats, resulted in growth arrest and abnormal leaf development. NbRae1 was mainly associated with the nuclear envelope during interphase, and NbRae1 deficiency caused accumulation of poly(A) RNA in the nuclei of leaf cells, suggesting defective mRNA export. In the shoot apex, depletion of NbRae1 led to reduced mitotic activities, accompanied by reduced cyclin-dependent kinase (CDK) activity and decreased expression of cyclin B1, CDKB1-1, and histones H3 and H4. The secondary growth of stem vasculature was also inhibited, indicating reduced cambial activities. Differentiated leaf cells of NbRae1 -silenced plants exhibited elevated ploidy levels. Immunolabeling in BY-2 cells showed that NbRae1 protein localized to mitotic microtubules and the cell plate-forming zone during mitosis, and recombinant NbRae1 directly bound to microtubules in vitro . Inhibition of NbRae1 expression in BY-2 cells using a β-estradiol-inducible RNAi system resulted in severe defects in spindle organization and chromosome alignment and segregation, which correlated with delays in cell cycle progression. Together, these results suggest that NbRae1 plays a dual role in mRNA export in interphase and in spindle assembly in mitosis.