TSA诱导MOLT-4细胞中p21WAF1/Cip-1表达的功能机制研究

为了研究去乙酰化酶抑制剂TSA诱导MOLT-4细胞周期阻滞和凋亡反应中p21^WAF1/Cip-1的表达及其功能，以组蛋白去乙酰化酶抑制剂TSA处理急性淋巴细胞白血病细胞系MOLT-4，流式细胞仪和细胞吖啶橙染色、瑞士染色检测细胞周期和凋亡，Western检测p21^WAF1/Cip-1。的表达。结果表明：组蛋白去乙酰化酶抑制剂TSA可有效的诱导MOLT-4细胞发生G2／M阻滞和凋亡，并且呈现明显的剂量效应关系和时间效应关系；在此周期阻滞与凋亡反应过程中，p21“…“‘川蛋白的表达水平在周期阻滞前快速增高，而在凋亡早期开始下降。p21^WAF1/Cip-1分子在细胞中的表达规律与TSA诱导的细胞G2／M阻滞和凋亡反应间存在明显的剂量效应关系和时间效应关系；蛋白酶体抑制剂MG—132提升p21^WAF1/Cip-1分子在细胞中的表达可以增进细胞的G2／M阻滞反应而延缓凋亡。结论：蛋白酶体途径参与TSA诱导的MOLT-4细胞周期阻滞向凋亡转换过程中p21^WAF1/Cip-1分子的降解调控；p21^WAF1/Cip-1在TSA诱导的MOLT4细胞G2／M阻滞和凋亡反应中起着重要调节作用。     

螺旋CT对椎弓根螺钉固定术各种数据的测量应用

目的 利用螺旋CT轴位及三维成像的表面遮盖法(surface shaded display,SSD)为临床提供椎弓根螺钉固定术术前水平面进钉角(transverse screw angle,TSA)、进钉轴线长度、椎弓根最小横径及椎弓根进钉间距等测量的精确数据。方法 取螺旋CT轴位椎弓根最宽层面及SSD图像进行TSA、进钉轴线长度、椎弓根最小横径、椎弓根进钉间距的测量。结果 该方法协助临床术前确定TsA，螺钉长度与大小的准备，进钉点及进钉间距。提高椎弓根螺钉固定术的质量及成功率。结论 该方法为临床术前提供TsA，螺钉长度、大小，进钉点及进钉间距等精确数据，是一种简单可靠的方法。     

乳腺癌中MAGE-A9和MAGE-A11基因的表达及表达机制的研究

【】 目的：探讨黑色素瘤抗原MAGE-A9和MAGE-A11在乳腺正常组织、乳腺良性病变、乳腺癌组织中的表达以及甲基化酶抑制剂5-氮杂-２"-脱氧胞苷(5-aza-CdR)和组蛋白去乙酰化酶抑制剂曲古抑菌素A(TSA)对MAGE-A9和MAGE-A11基因表达的影响。方法：应用RT-PCR检测60例乳腺正常组织、60例乳腺良性病变及60例乳腺癌组织中MAGE-A9和MAGE-A11 mRNA的表达,分析其与乳腺癌患者临床病理学特征的关系。乳腺癌细胞MCF-7、MDA-MB-231经5-aza-CdR 和 TSA 单独或联合作用后,应用RT-PCR法检测细胞中 MAGE-A9和MAGE-A11 mRNA 表达的变化。结果：乳腺癌组织中MAGE-A9 mRNA阳性表达率为45%(27/60),MAGE-A11 mRNA阳性表达率为66.7%(40/60),而乳腺正常组织及良性病变中均未发现MAGE-A9和MAGE-A11 mRNA的表达。 MAGE-A9和MAGE-A11 mRNA的表达与乳腺癌患者的年龄、病理类型、临床分期、肿瘤大小、淋巴转移、瘤栓及孕激素受体的表达均无明显关系(P ＞0.05),与人表皮生长因子受体2(HER-2)和雌激素受体(ER)的表达有关(P＜0.05)。未经药物处理的MCF-7、MDA-MB-231细胞未见MAGE-A9和MAGE-A11 mRNA表达。单用5-aza-CdR后可见MAGE-A9和MAGE-A11基因重新表达,联合应用5-aza-CdR和TSA使MAGE-A9和MAGE-A11基因表达进一步增加(P＜0.05)。TSA单独作用对基因表达没有影响(P＞0.05)。结论：乳腺癌组织中MAGE-A9和MAGE-A11 mRNA的表达与ER 和HER-2的表达有关,5-aza-CdR单用或联合应用TSA可诱导不表达MAGE-A9和MAGE-A11 mRNA的细胞重新表达,说明DNA的去甲基化和组蛋白乙酰化是调控人类肿瘤细胞MAGE的表达的重要机制。     

HDAC activity is required for BDNF to increase quantal neurotransmitter release and dendritic spine density in CA1 pyramidal neurons

Molecular mechanisms involved in the strengthening and formation of synapses include the activation and repression of specific genes or subsets of genes by epigenetic modifications that do not alter the genetic code itself. Chromatin modifications mediated by histone acetylation have been shown to be critical for synaptic plasticity at hippocampal excitatory synapses and hippocampal-dependent memory formation. Considering that brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity and behavioral adaptations, it is not surprising that regulation of this gene is subject to histone acetylation changes during synaptic plasticity and hippocampal-dependent memory formation. Whether the effects of BDNF on dendritic spines and quantal transmitter release require histone modifications remains less known. By using two different inhibitors of histone deacetylases (HDACs), we describe here that their activity is required for BDNF to increase dendritic spine density and excitatory quantal transmitter release onto CA1 pyramidal neurons in hippocampal slice cultures. These results suggest that histone acetylation/deacetylation is a critical step in the modulation of hippocampal synapses by BDNF. Thus, mechanisms of epigenetic modulation of synapse formation and function are novel targets to consider for the amelioration of symptoms of intellectual disabilities and neurodegenerative disorders associated with cognitive and memory deficits.     

DNA methylation signatures of the AIRE promoter in thymic epithelial cells, thymomas and normal tissues

Mutations in the AIRE gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), which is associated with autoimmunity towards several peripheral organs. The AIRE protein is almost exclusively expressed in medullary thymic epithelial cells (mTEC) and CpG methylation in the promoter of the AIRE gene has been suggested to control its tissue-specific expression pattern. We found that in human AIRE-positive medullary and AIRE-negative cortical epithelium, the AIRE promoter is hypomethylated, whereas in thymocytes, the promoter had high level of CpG methylation. Likewise, in mouse mTECs the AIRE promoter was uniformly hypomethylated. In the same vein, the AIRE promoter was hypomethylated in AIRE-negative thymic epithelial tumors (thymomas) and in several peripheral tissues. Our data are compatible with the notion that promoter hypomethylation is necessary but not sufficient for tissue-specific regulation of the AIRE gene. In contrast, a positive correlation between AIRE expression and histone H3 lysine 4 trimethylation, an active chromatin mark, was found in the AIRE promoter in human and mouse TECs.     

Binding affinity in drug design: experimental and computational techniques

ABSTRACT

Introduction: In pharmaceutical design where future drugs are developed by targeting a specific chosen protein, the evaluation of ligand affinity is crucial. For this very purpose are a multitude of diverse methods which are continuously being improved, which, in turn, makes it difficult to choose which techniques to use in practice.

Areas covered: In this review, the authors discuss both experimental and computational approaches for affinity evaluation. Basic principles, general limitations and advantages, as well as main areas of application in drug discovery, are overviewed for some of the most popular ligand binding assays. The authors further provide a guide to affinity predictions, collectively covering several techniques that are used in the first stages of rational drug design.

Expert opinion: All affinity estimation methods have limitations and advantages that partially overlap and complement one another. Some of the suggested best practices include cross-verification of data using at least two different techniques and careful data interpretation.     

曲古抑菌素A增强细胞因子诱导的杀伤细胞对胰腺癌细胞株杀伤效果的体外研究

目的：研究细胞因子诱导的杀伤（CIK）细胞对胰腺癌细胞株的杀伤效果;探索曲古抑菌素A（TSA）增强其杀伤效果的机制。方法将胰腺癌细胞株分为2组。一组经100 mmol/L;200 mmol/L浓度的TSA处理24 h,另外一组为空白对照。分别使用流式细胞仪测定两组胰腺癌细胞株的MICA/B表达及使用乳酸脱氢酶法测定CIK细胞对两组胰腺癌细胞株的杀伤率。双变量相关性分析胰腺癌细胞株MICA/B的表达与CIK细胞对胰腺癌细胞株杀伤率的关系。结果经TSA处理24 h后的胰腺癌细胞株,其MICA/B表达相较对照组有明显的升高（P＝0.000）,有统计学差异;在不同靶效比的情况下,CIK 细胞对经 TSA处理24 h后的胰腺癌细胞株的杀伤率有显著的提高（P＝0.000）;而相关性分析显示,CIK细胞对胰腺癌细胞株杀伤率的升高与胰腺癌细胞株MICA/B的表达呈正相关（P＜0.01）。结论 TSA可显著调高胰腺癌细胞株的MICA/B表达（P＜0.01）,从而增强CIK细胞对胰腺癌细胞株的杀伤。     

Reversal of ER-β silencing by chromatin modifying agents overrides acquired tamoxifen resistance

The purpose of this work is to determine the molecular mechanisms underlying tamoxifen resistance. We show here that ER-β is epigenetically silenced in a cell line with acquired tamoxifen resistance (MCF-7/TAM-R) and this could be reversed by 5-AZA-deoxycytidine (5-AZA) and trichostatin-A (TSA) pre-treatment. Subsequent treatment with 4-hydroxy-tamoxifen (4-OHT) induced ER-β nuclear translocation, upregulated pS2 and p21 levels and reduced cell viability. Transfection with an ER-β expression vector sensitized MCF-7/TAM-R cells to the growth inhibitory and pro-apoptotic effects of 4-OHT, indicating that ER-β re-expression alone is sufficient to restore sensitivity to tamoxifen. This novel finding reveals that ER-β is fundamental in overcoming acquired tamoxifen resistance and provides insights for new therapeutic protocols against breast cancer.