TRF1、TRF2和POT1基因mRNA在前列腺癌组织中的表达

目的:探究端粒重复序列结合蛋白质1(TRF1)、TRF2和端粒保护蛋白(POT1)基因mRNA在前列腺癌(PCa)组织中的表达。方法:收集46例PCa患者肿瘤中心组织(中心组织组)和35例前列腺增生(BPH)患者BPH组织(BPH组织组),提取总RNA,逆转录成cDNA,采用定量PCR测定TRF1、TRF2和POT1在中心组织组和BPH组织基因mRNA的表达,采用基因mRNA所得CT值与β-actin mRNA所得CT值之差(△CT值)表示,并进行差异性分析。结果:中心组织组TRF1的△CT值高于BPH组织组,差异有统计学意义(Z=-3.469,P=0.001);TRF2的△CT值分别为8.49(5.75,10.21)和8.16(6.28,9.75),差异无统计学意义(Z=-1.719,P=0.086);中心组织组POT1的△CT值高于BPH组织组,差异有统计学意义(t=-18.48,P=0.000)。结论:TRF1和POT1在PCa肿瘤组织中的表达升高,说明TRF1和POT1的高表达可能与PCa的发生和发展有关,但TRF2在PCa肿瘤中心组织和BPH组织中的表达没有表现出差异,有待进一步研究。     

组蛋白乙酰化在二烯丙基二硫诱导MGC803细胞分化中的作用

探讨二烯丙基二硫(DADS)诱导人胃癌MGC803细胞分化过程中组蛋白乙酰化状态的改变情况。运用形态学方法及成瘤实验观察DADS诱导MGC803细胞分化,应用Western印迹观察DADS诱导MGC803细胞分化与其调控细胞组蛋白乙酰化水平和相关p21WAF1的关系。形态学观察结果显示,30mg/LDADS处理MGC803细胞24h后,细胞异型性明显减少,且经裸鼠成瘤实验证实,处理后的细胞均未在裸鼠体内形成肿瘤;Western印迹显示,30mg/LDADS处理细胞12h后,其组蛋白H3乙酰化程度明显升高,与未处理组比较增加了38%(P<0.05);H4乙酰化程度无明显改变。用15、30、60mg/LDADS处理细胞12、24h后,p21WAF1均较对照组升高,以30mg/LDADS处理24h升高最显著。研究结果表明,DADS可诱导MGC803细胞分化,其作用可能与增加核组蛋白乙酰化水平及p21WAF1表达有关。     

毛茛有效部位D1对人胃癌细胞MGC803细胞增殖和凋亡的作用及其机制的初步研究

采用AlamarBlue和瑞-姬氏染色方法检测毛茛有效部位D1对人胃癌细胞MGC803增殖的作用;PI染色法观察D1对MGC803细胞周期的影响;半定量RT-PCR法检测D1对MGC803细胞周期相关基因表达的作用;用DAPI染色方法和细胞色素C免疫荧光法观察D1对胃癌细胞MGC803凋亡的影响。结果显示,毛茛有效部位D1对胃癌细胞MGC803表现出较好的增殖抑制作用,且24、48和72 h的半数抑制浓度分别为126.89、74.81、68.72μg/mL;同时D1对细胞周期和周期相关基因的表达无明显影响,且D1能促使细胞色素C释放到细胞胞浆诱导细胞凋亡。     

P53与TRF1、TRF2的体外结合及P53结合功能区的分析

通过分析端粒结合蛋白TRF1、TRF2与P5 3的体外结合 ,探讨P5 3 端粒途径调节细胞生命活动的分子机制 .GST和 4种人P5 3 GST融合蛋白经大肠杆菌表达、谷胱甘肽 SepharoseTM4B纯化后 ,进行SDS PAGE和考马斯亮篮染色 .人P5 3包括野生型 (1～ 393)、C端缺失体P5 3N5 (2～ 2 93)、N端缺失体P5 32C(95～ 393)、单个氨基酸突变体P5 3R175H(175R→H) .各纯化蛋白的分子量与预计的完全一致 ,且纯化率达 90 %以上 .将纯化的GST和P5 3 GST融合蛋白与人乳腺癌细胞MCF 7细胞蛋白进行体外结合反应 ,Western印迹检测反应物中P5 3和TRF1、TRF2的结合 .野生型P5 3和P5 3 R175H均能沉淀MCF 7中的TRF1、TRF2 ,且结合力相似 ,而单独的GST则无沉淀TRF1、TRF2的作用 .与野生型P5 3和P5 3R175H相比 ,P5 32C与TRF1、TRF2的结合力明显增加 ,P5 3N5与TRF1、TRF2的结合力大大减弱 .表明P5 3和TRF1、TRF2可以进行直接而特异的体外结合 ,且它们的结合为P5 3C端 (2 93～ 393)结构域依赖性 .P5 3和TRF1、TRF2这种结构域依赖性的结合可能与端粒动态变化所诱导的细胞活动有关 .     

正确认识Th1—Th2细胞在传染病中的作用

本文对Ｔｈ１及Ｔｈ２细胞在传染病中的作用提出了置疑，认为Ｔｈ１应符保护细胞内寄生病原，Ｔｈ２应答保护细胞外寄生病原体的说法是教条，严重地影响研究的进展。作者认为不应把复杂的免疫学现象简单化。     

Th1/Th2细胞在心肌梗死中的作用

Th1/Th2细胞因子平衡对于维持机体的正常免疫状态是重要因素,其平衡的紊乱与很多疾病密切相关,如感染性疾病、自身免疫性疾病、肿瘤、心血管疾病等.在心肌梗死的发生发展过程中存在炎症反应,病变的心肌细胞及其他炎症细胞均可产生大量炎症细胞因子和炎症介质,有研究证实这些细胞因子影响心肌重构.近年来关于Th1/Th2细胞因子的平衡与心血管疾病的关系已成为研究的重点.急性心肌梗死的发作与免疫应答的关系越来越引起人们的关注.本文就Th1/rh2细胞在心肌梗死中的作用做一简要综述.     

TFCP2L1在子宫内膜癌细胞增殖及侵袭中的作用

摘要 目的：研究人子宫内膜癌中TFCP2L1的表达情况以及分析TFCP2L1对子宫内膜癌细胞增殖及迁移能力的影响。方法：（1）通过TCGA 及GTEx数据库分析子宫内膜癌中TFCP2L1的表达水平及患者生存期。采用Western blot验证正常子宫内膜上皮细胞与多种子宫内膜癌细胞系中TFCP2L1的表达情况。（2）使用CRISPR-Cas9技术敲除Ishikawa细胞系的TFCP2L1,并用流式分选技术筛选单个细胞进行培养,形成单克隆细胞系,以此来研究TFCP2L1对子宫内膜癌的细胞周期和细胞增殖的影响。通过 Western blot 及细胞免疫荧光检测细胞周期相关蛋白的表达,检测细胞增殖情况,采用平板克隆实验及CCK8实验。（3）通过Transwell小室及划痕实验对侵袭和转移能力进行检测。结果：TCGA 及GTEx数据库分析发现TFCP2L1在子宫内膜癌中高表达且与肿瘤患者预后不良相关。敲除TFCP2L1后,Ki67、Cyclin D1 和 Cyclin D2的蛋白水平显著下调,CCK8及平板克隆实验结果表明,敲除TFCP2L1能够显著降低子宫内膜癌细胞的增殖能力。划痕实验及Transwell侵袭实验结果表明敲除TFCP2L1的子宫内膜癌细胞侵袭迁移能力均减弱。结论：本研究证明了TFCP21L1是子宫内膜癌的促癌因子。TFCP2L1的高表达可能与子宫内膜癌预后不良相关。敲除TFCP2L1可以抑制子宫内膜癌的侵袭和转移。     

特异AT序列结合蛋白-1促进胰岛素样生长因子 结合蛋白-2在K562细胞中的表达

探讨过表达特异AT序列结合蛋白-1 ( special AT-rich sequence binding protein ,SATB1)核基质结合区（MAR）结合蛋白对胰岛素样生长因子结合蛋白-2（IGFBP2）基因表 达的影响,并对其影响机制进行初步探索．首先用脂质体将SATB1的真核表达载体pcDNA3.1-SATB1转染至K562细胞,通过6周G418的筛选获得阳性克隆,RT-PCR、实时PCR及Western 印迹验证过表达情况,对阳性克隆细胞中IGFBP2的表达用RT-PCR、实时PCR及Western 印迹方法进行检测;然后用RNAi的方法干扰阳性细胞中SATB1 的表达后,同样用上述3种方法再次检测IGFBP2的表达状况;用生物信息学方法对IGFBP2基因进行MAR序列与SATB1结合位点搜索分析,寻找SATB1影响IGFBP2基因表达的机制．结果显示,在稳定转染的情况下,实验组K562-SATB1细胞与转染空载体pcDNA3.1的K562-3.1细胞和未转染细胞K562相比,IGFBP2 mRNA水平上调了近7倍,而蛋白水平变化不明显．RNA干扰后,IGFBP2的表达在mRNA水平也相应下调,蛋白水平的变化同样不明显．通过生物信息学分析发现,IGFBP2第1个内含子中可能存在2. 5 kb MAR样序列,且MAR样序列上存在多个SATB1的潜在结合位点．综上所述,过表达SATB1可以使K562细胞中IGFBP2 mRNA表达水平提高,而且其调控机制可能与SATB1直接和IGFBP2基因中的MAR样序列结合有关．     

COX-2 与mPGES-1 在肾透明细胞癌中的表达及临床意义

目的:探讨环氧合酶-2(COX-2)和膜结合型前列腺素E2合成酶1(mPGES-1)在肾透明细胞癌组织中的表达及临床意义。方法:采用免疫组化SP法分别检测49例肾透明细胞癌组织标本和21例正常肾组织标本中COX-2和mPGES-1的表达。结果:COX-2在正常肾组织中的阳性表达率为4.8%,在肾透明细胞癌组织中的阳性表达率为53.1%(P<0.05);mPGES-1在正常肾组织中的阳性表达率为4.8%,在肾透明细胞癌组织中的阳性表达率为40.8%(P<0.05);COX-2和mPGES-1的高表达均与肾透明细胞癌的病理分级和临床分期无相关性(P>0.05);COX-2和mPGES-1在肾透明细胞癌中的表达呈正相关(P<0.05),r=0.5。结论:COX-2和mPGES-1在肾透明细胞癌发生及发展过程中共同发挥重要作用;COX-2和mPGES-1可能成为肾透明细胞癌新的治疗靶点。     

转谷氨酰胺酶2抑制H1亚型流感病毒在MDCK细胞中的增殖

组织转谷酰胺酶(transglutaminase 2,TGM2)是一种普遍存在的多功能蛋白,与不同细胞的粘附和肿瘤形成有关.有证据表明,TGM2参与了宿主细胞与病毒间的相互作用,但是对于流感病毒在细胞内增殖的影响还未有报道.为了探究MDCK细胞中TGM2对H1N1亚型流感病毒增殖的影响,本研究构建了TGM2过表达和敲除...     

人端粒结合蛋白TRF1的克隆、表达和抗体制备

利用RT-PCR技术,从HeLa细胞的cDNA文库中扩增到人端粒结合蛋白1(hTRF1)基因编码区序列,克隆至pUCm-T载体,测序正确后,构建带His₆-tag原核表达载体pET-28c-TRF1,经IPTG诱导表达的His₆-TRF1融合蛋白分子量约为65kD,Western-blot证实表达产物可特异地与TRF1抗体sc-6165结合。用Ni^2＋NTA胶亲和层析纯化可得到电泳均一的融合蛋白,免疫新西兰纯种大白兔,获得特异性好的多克隆抗体,该抗体可用于免疫荧光染色和Western-blot方法检测哺乳动物细胞内源性的TRF1分子。     

Silencing of Bcl-XL Expression in Human MGC-803 Gastric Cancer Cells by siRNA

To investigate the inhibitory effect of the Bcl-XL small interfering RNA(siRNA)on BcI-XLgene expression in the human gastric cancer cell line MGC-803,green fluorescent protein(GFP)siRNAwas constructed and transfected into MGC-803 ceils,together with GFP expression vector pTrace SV40.GFP expression levels were observed using fluorescence microscopy.Bcl-XL siRNA and negative siRNAwere then constructed and stably transfected into MGC-803 cells.RT-PCR and immunofluorescence wereused to detect the expression of Bcl-XL.Spontaneous apoptosis was detected by acridine orange(AO)andflow cytometry.Results were as follows:(1)48 h after GFP expression vector and GFP siRNA co-transfection,the expression level of GFP in the GFP siRNA group was much lower than the negative siRNA group,according to fluorescence microscopy results.The mRNA and protein levels of Bcl-XL in Bcl-XL siRNAstable transfectants were reduced to almost background level compared with negative siRNA transfectantsor untreated cells.(2)Changes in nucleus morphology was observed by AO staining nucleic and flowcytometry analysis,which showed that stable Bcl-XL siRNA transfectants have an increased spontaneousapoptosis (21.17%+1.26% vs.1.19%+0.18% and 1.56%+0.15% respectively,P<0.05 vs.negative siRNAor untreated control),siRNA targeting GFP or Bcl-XL genes can specifically suppress GFP or BcI-XLexpression in MGC-803 cells,and Bcl-XL siRNA can increase spontaneous apoptosis.Bcl-XL siRNA maybe a beneficial agent against human gastric adenocarcinoma.     

Structure,dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD⁺ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.     

Cell cycle-dependent 3D distribution of telomeres and telomere repeat-binding factor 2 (TRF2) in HaCaT and HaCaT-myc cells

Telomeres are specialized structures at the ends of the chromosomes that, with the help of proteins--such as the telomere repeat-binding factor TRF2 -, form protective caps which are essential for chromosomal integrity. Investigating the structure and three-dimensional (3D) distribution of the telomeres and TRF2 in the nucleus, we now show that the telomeres of the immortal HaCaT keratinocytes are distributed in distinct non-overlapping territories within the inner third of the nuclear space in interphase cells, while they extend more widely during mitosis. TRF2 is present at the telomeres at all cell cycle phases. During mitosis additional TRF2 protein concentrates all around the chromosomes. This change in staining pattern correlates with a significant increase in TRF2 protein at the S/G2 transition as seen in Western blots of synchronized cells and is paralleled by a cell cycle-dependent regulation of TRF2 mRNA, arguing for a specific role of TRF2 during mitosis. The distinct territorial localization of telomeres is abrogated in a HaCaT variant that constitutively expresses c-Myc--a protein known to contribute to genomic instability. These cells are characterized by overlapping telomere territories, telomeric aggregates (TAs), that are accompanied by an overall irregular telomere distribution and a reduced level in TRF2 protein. These TAs which are readily detectable in interphase nuclei, are similarly present in mitotic cells, including cells in telophase. Thus, we propose that TAs, which subsequently also cluster their respective chromosomes, contribute to genomic instability by forcing an abnormal chromosome segregation during mitosis.     

Chromosomal distribution of interstitial telomeric sequences as signs of evolution through chromosome fusion in six species of the giant water bugs (Hemiptera,Belostoma)

Tandem arrays of TTAGG repeats show a highly conserved location at the telomeres across the phylogenetic tree of arthropods. In giant water bugs Belostoma, the chromosome number changed during speciation by fragmentation of the single ancestral X chromosome, resulting in a multiple sex chromosome system. Several autosome–autosome fusions and a fusion between the sex chromosome pair and an autosome pair resulted in the reduced number in several species. We mapped the distribution of telomeric sequences and interstitial telomeric sequences (ITSs) in Belostoma candidulum (2n = 12 + XY/XX; male/female), B. dentatum (2n = 26 + X₁X₂Y/X₁X₁X₂X₂), B. elegans (2n = 26 + X₁X₂Y/X₁X₁X₂X₂), B. elongatum (2n = 26 + X₁X₂Y/X₁X₁X₂X₂), B. micantulum (2n = 14 + XY/XX), and B. oxyurum (2n = 6 + XY/XX) by FISH with the (TTAGG)_n probes. Hybridization signals confirmed the presence of TTAGG repeats in the telomeres of all species examined. The three species with reduced chromosome numbers showed additional hybridization signals in interstitial positions, indicating the occurrence of ITS. From the comparison of all species here analyzed, we observed inverse relationships between chromosome number and chromosome size, and between presence/absence of ITS and chromosome number. The ITS distribution between these closely related species supports the hypothesis that several telomere–telomere fusions of the chromosomes from an ancestral diploid chromosome number 2n = 26 + XY/XX played a major role in the karyotype evolution of Belostoma. Consequently, our study provide valuable features that can be used to understand the karyotype evolution, may contribute to a better understanding of taxonomic relationships, and also elucidate the high plasticity of nuclear genomes at the chromosomal level during the speciation processes.     

Telomere elongation by a mutant tankyrase 1 without TRF1 poly(ADP-ribosyl)ation

Telomeres are the capping structures of the eukaryotic chromosome ends. Tankyrase 1 is a poly(ADP-ribose) polymerase that elongates telomeres in a telomerase-dependent manner. This function of tankyrase 1 is mediated by down-regulation of TRF1, a negative regulator of telomere access to telomerase. Namely, tankyrase 1 poly(ADP-ribosyl)ates (PARsylates) TRF1, which in turn dissociates TRF1 from telomeres. The resulting telomeres become better substrates for telomerase-mediated DNA extension. Tankyrase 1 has five independent TRF1 binding sites, ARC (ANK repeat cluster) I to V. Among them, the most C-terminal ARC V is required for TRF1 PARsylation and its release from telomeres. By contrast, functional significance of other four ARCs remains elusive. In this study, we generated a mutant tankyrase 1 that had inactive ARC IV and lacked ARC V but elongated telomeres without TRF1 PARsylation. Consistent with the failure in PARsylation, this mutant only marginally released TRF1 from telomeres. Still, it decreased telomere binding of POT1, a downstream effector of TRF1-mediated telomere length control, and elongated the telomeric 3'-overhang as the wild-type tankyrase 1 did. Thus even without TRF1 PARsylation, this mutant tankyrase 1 seemed to loosen the closed structure of the telomeric heterochromatin. These findings suggest a new role for multiple ARCs in telomere extension by tankyrase 1.     

Role of the Alkali Labile Sites,Reactive Oxygen Species and Antioxidants in DNA Damage Induced by Methylated Trivalent Metabolites of Inorganic Arsenic

In the last decade arsenic metabolism has become an important matter of discussion. Methylation of inorganic arsenic (iAs) to monomethylarsonic acid (MMA^V) and dimethylarsinic acid (DMA^V) is considered to decrease arsenic toxicity. However, in addition to these pentavalent metabolites, the trivalent metabolites monomethylarsonous (MMA^III) and dimethylarsinous acid (DMA^III) have been identified recently as intermediates in the metabolic pathway of arsenic in cultured human cells. To examine the role of oxidative damage in the generation of DNA strand breaks by methylated trivalent arsenic metabolites, we treated human lymphocytes with both metabolites at non-cytotoxic concentrations. We further tested whether these effects are sensitive to modulation by the antioxidants ascorbate (Vitamin C) and selenomethionine (Se-Met). Both trivalent metabolites produced oxidative stress related DNA damage, consisting of single strand breaks and alkali-labile sites, with MMA^III being more potent at low concentrations than DMA^III. Neither MMA^III nor DMA^III induced DNA-double strand breaks. The oxidative stress response profiles of the metabolites were parallel as determined by lipid peroxidation induction. MMA^III induced peroxidation from the lowest concentration tested, while effects of DMA^III were apparent only at concentrations above 10 μM. The antioxidant Se-Met exhibited a more pronounced inhibition of trivalent arsenic metabolite-induced oxidative-DNA damage than did vitamin C. The present findings suggest that DNA damage by methylated trivalent metabolites at non-cytotoxic concentrations may be mediated by a mix of reactive oxygen and nitrogen oxidized species.     

Mitotic perturbations induced by Nek2 overexpression require interaction with TRF1 in breast cancer cells

NIMA-related kinase 2 (Nek2), a serine–threonine protein kinase, plays a major role in mitotic progression, including timing of mitotic entry, chromatin condensation, spindle organization, and cytokinesis. Nek2 overexpression results in premature centrosome separation, while kinase death Nek2 mutant expression or Nek2-depleted cells lead to centrosome separation failure. In addition, it has been revealed that telomeric repeat binding factor 1 (TRF1) interacts directly with Nek2. TRF1 not only regulates telomere length, but is also associated with cell cycle regulation. However, the interactions and correlations between Nek2 and TRF1 are far from clear. Here, we show that mitotic aberrations through Nek2 overexpression are likely to require TRF1. Our results demonstrate that Nek2 directly binds and phosphorylates TRF1 through multiple sites on TRF1. Nek2 overexpression in breast cancer cells, MDA-MB-231 and MCF7, results in increased numbers of centrosomes and multinucleated cells, which leads to cytokinetic failure and aneuploidization. Additionally, TRF1 depletion by siRNA prevents the phenomenon of unaligned chromosomes by Nek2 overexpression during metaphase. Concurrent Nek2 overexpression and TRF1-depleted cells demonstrated ≤ 2 centrosomes per cell, similar to mock plasmid and negative control siRNA-transfected cells. Interestingly, when exogenous TRF1 was added back in Nek2-overexpressed cells with endogenous TRF1 depletion, cells had re-induced cytokinetic failure. Therefore, we propose that TRF1 is required for overexpressed Nek2 to trigger abnormal mitosis and chromosomal instability.