亚硒酸钠对肝细胞L-02端粒酶活性和端粒长度的作用

通过研究硒对端粒酶活性和端粒长度的作用 ,探讨硒抗衰老的生物学机制。实验以人肝细胞株L 0 2为研究对象 ,分别补充 0 .5和 2 .5 μmol L亚硒酸钠 ,采用端粒重复序列扩增 焦磷酸根酶联发光法、逆转录聚合酶链式反应法及流式荧光原位杂交法 ,分别检测细胞的端粒酶活性、人端粒酶逆转录酶催化亚基基因 (hTERT)的表达及端粒长度的变化。结果表明 :常规培养的肝细胞株L 0 2的端粒酶活性和hTERT基因表达水平均较低。补充 0 .5和2 .5 μmol L亚硒酸钠三周后细胞生长状况良好、端粒酶活性和hTERT基因表达水平显著性增高 ,且呈一定的剂量 效应关系。细胞补充亚硒酸钠四周后端粒长度显著增长。说明营养浓度的亚硒酸钠可通过提高端粒酶活性和增长端粒长度来减缓L 0 2肝细胞衰老、延长细胞寿命。     

Telomere Length Dynamics in Telomerase-Positive Immortal Human Cell Populations

It has been proposed that the progressive shortening of telomeres in somatic cells eventually results in senescence. Previous experiments have demonstrated that many immortal cell lines have acquired telomerase activity leading to stabilization of telomere length. Telomere dynamics and telomerase activity were examined in the telomerase-positive immortal cell lines HeLa and 293 and subclones derived from them. A mass culture of HeLa cells had a stable mean telomere length over 60 population doublings (PD)in vitro.Subclones of this culture, however, had a range of mean telomere lengths indicating that telomeric heterogeneity exists within a population with a stable mean telomere length. Some of the subclones lacked detectable telomerase activity soon after isolation but regained it by PD 18, suggesting that at least some of the variation in telomere length can be attributed to variations in telomerase activity levels. 293 subclones also varied in telomere length and telomerase activity. Some telomerase-positive 293 subclones contained long telomeres that gradually shortened, demonstrating that factors other than telomerase also act to modulate telomere length. Fluctuations in telomere length in telomerase-positive immortalized cells may contribute to chromosomal instability and clonal evolution.     

端粒结合蛋白与端粒长度调控

真核细胞端粒DNA序列的丢失与细胞的衰老及凋亡有关.端粒酶的激活可维持端粒长度并使细胞获得无限增殖的能力.端粒结合蛋白则可能通过调节端粒酶或其他相关因子的行为参与对端粒长度的调控.近年有关端粒结合蛋白的研究取得了突破性进展并在此基础上建立了端粒长度调控模型.     

The N Terminus of the OB Domain of Telomere Protein TPP1 Is Critical for Telomerase Action

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地高辛标记法检测人源细胞系端粒长度

目的:建立一种灵敏的、非同位素标记的端粒长度检测方法,并用于人源细胞系的肿瘤研究。方法:以地高辛标记寡核苷酸(TTAGGG)3为探针,对基因组DNA进行Southern印迹检测,经CDP-Star显色后与DNA标准分子量比较,利用图像分析系统计算端粒长度。结果:通过严格控制探针的工作浓度(1∶3000稀释)、DNA上样量(1.5～2.5μg)、杂交温度(42±1℃)等主要影响因素,获得了比较好的杂交条带。结论:建立了一种稳定、可靠、低背景的地高辛标记检测端粒长度的方法;对正常细胞、永生化细胞和肿瘤细胞进行了端粒长度的检测和对比分析。     

高等植物端粒和端粒酶的研究进展

端粒是构成真核生物线状染色体末端重要的DNA-蛋白质复合结构,DNA由简单的串联重复序列组成。它的合成由一个特殊的具有反转录活性的核糖核蛋白-端粒酶完成。端粒对染色体、整个生物基因组,甚至对细胞的稳定都具有重要意义。本文就植物端粒、端粒酶、端粒结合蛋白,以及端粒变化、端粒酶在植物生长发育中的调节作一概述。     

Telomere uncapping during in vitro T-lymphocyte senescence

Normal lymphocytes represent examples of somatic cells that are able to induce telomerase activity when stimulated. As previously reported, we showed that, during lymphocyte long-term culture and repeated stimulations, the appearance of senescent cells is associated with telomere shortening and a progressive drop in telomerase activity. We further showed that this shortening preferentially occured at long telomeres and was interrupted at each stimulation by a transitory increase in telomere length. In agreement with the fact that telomere uncapping triggers lymphocyte senescence, we observed an increase in γ-H2AX and 53BP1 foci as well as in the percentage of cells exhibiting DNA damage foci in telomeres. Such a DNA damage response may be related to the continuous increase of p16 ^ink4a upon cell stimulation and cell aging. Remarkably, at each stimulation, the expression of shelterin genes, such as hTRF1 , hTANK1 , hTIN2 , hPOT1 and hRAP1 , was decreased. We propose that telomere dysfunction during lymphocyte senescence caused by iterative stimulations does not only result from an excessive telomere shortening, but also from a decrease in shelterin content. These observations may be relevant for T-cell biology and aging.     

动物克隆中的端粒和端粒酶

端粒是染色体上的一种重要结构,对维持染色体的稳定性起重要作用。核移植后,端粒长度和端粒酶活性的变化是重要的核重编程事件。不同种类的动物和供体细胞核移植后,在端粒长度的变化上存在一些差异,反映了重编程程度的不同。核移植后,在克隆囊胚中存在高水平的端粒酶活性,克隆动物的端粒长度延长,可能是由于克隆过程中端粒酶基因的重编程的缘故。     

端粒和端粒酶与衰老研究

衰老是一种多因素的复合调控过程,表现为染色体端粒长度的改变、DNA损伤、DNA的甲基化和细胞的氧化损伤等,并已形成了许多学说,而端粒学说成为衰老研究的热点之一.对与衰老紧密相关的因素———端粒、端粒酶的结构及其与衰老关系的研究进展进行综述,阐明对端粒—端粒酶的作用将会在抗衰老方面有着十分重要的理论价值及实际意义.     

Loss of Human TGS1 Hypermethylase Promotes Increased Telomerase RNA and Telomere Elongation

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Telomere Structure,Replication and Length Maintenance

Telomeres are the termini of linear eukaryotic chromosomes consisting of tandem repeats of DNA and proteins that bind to these repeat sequences. Telomeres ensure the complete replication of chromosome ends, impart protection to ends from nucleolytic degradation, end-to-end fusion, and guide the localization of chromosomes within the nucleus. In addition, a combination of genetic, biochemical, and molecular biological approaches have implicated key roles for telomeres in diverse cellular processes such as regulation of gene expression, cell division, cell senescence, and cancer. This review focuses on recent advances in our understanding of the organization of telomeres, telomere replication, proteins that bind telomeric DNA, and the establishment of telomere length equilibrium.     

端粒与端粒酶的研究——解读2009年诺贝尔生理学或医学奖

三位美国科学家(Elizabeth H. Blackburn, Carol W. Greider 和Jack W. Szostak)因发现“端粒和端粒酶是如何保护染色体的”获得了2009年的诺贝尔生理学或医学奖．端粒是染色体末端的特殊结构,对染色体有保护作用,而端粒酶能合成端粒,使得端粒的长度和结构得以稳定．研究发现,端粒长度和端粒酶活性与细胞的寿命以及很多疾病发生直接相关．随着研究的不断深入,实现合理控制端粒的长度和端粒酶活性成为可能,这将有助于攻克医学领域“癌症、特定遗传病和衰老”三个重要领域的难题,有望研究开发出潜在的新疗法．     

哺乳动物早期胚胎端粒和端粒酶重编程

端粒位于真核染色体末端,是稳定染色体末端的重要元件。端粒酶(TER)是一种特殊的细胞核糖核蛋白(RNP)反转录酶(RT),其核心酶包括蛋白亚基和RNA元件。在DNA复制过程中的端粒丢失可以被有活性的端粒酶修复回来。哺乳动物端粒酶在发育中受调控,端粒的重编程可能是由于早期胚胎不同时期的端粒酶活性而造成的。因此,研究端粒和端粒酶重编程在早期胚胎发育中是非常重要的。该文综述了端粒和端粒酶的结构和功能,及其与哺乳动物早期胚胎发育的关系,并在此基础上展望了端粒和端粒酶在克隆动物胚胎发育的基础研究。     

The Principal Role of Ku in Telomere Length Maintenance Is Promotion of Est1 Association with Telomeres

Telomere length is tightly regulated in cells that express telomerase. The Saccharomyces cerevisiae Ku heterodimer, a DNA end-binding complex, positively regulates telomere length in a telomerase-dependent manner. Ku associates with the telomerase RNA subunit TLC1, and this association is required for TLC1 nuclear retention. Ku–TLC1 interaction also impacts the cell-cycle-regulated association of the telomerase catalytic subunit Est2 to telomeres. The promotion of TLC1 nuclear localization and Est2 recruitment have been proposed to be the principal role of Ku in telomere length maintenance, but neither model has been directly tested. Here we study the impact of forced recruitment of Est2 to telomeres on telomere length in the absence of Ku’s ability to bind TLC1 or DNA ends. We show that tethering Est2 to telomeres does not promote efficient telomere elongation in the absence of Ku–TLC1 interaction or DNA end binding. Moreover, restoration of TLC1 nuclear localization, even when combined with Est2 recruitment, does not bypass the role of Ku. In contrast, forced recruitment of Est1, which has roles in telomerase recruitment and activation, to telomeres promotes efficient and progressive telomere elongation in the absence of Ku–TLC1 interaction, Ku DNA end binding, or Ku altogether. Ku associates with Est1 and Est2 in a TLC1-dependent manner and enhances Est1 recruitment to telomeres independently of Est2. Together, our results unexpectedly demonstrate that the principal role of Ku in telomere length maintenance is to promote the association of Est1 with telomeres, which may in turn allow for efficient recruitment and activation of the telomerase holoenzyme.     

Telomerase enzymatic component hTERT shortens long telomeres in human cells

Telomere lengths are tightly regulated within a narrow range in normal human cells. Previous studies have extensively focused on how short telomeres are extended and have demonstrated that telomerase plays a central role in elongating short telomeres. However, much about the molecular mechanisms of regulating excessively long telomeres is unknown. In this report, we demonstrated that the telomerase enzymatic component, hTERT, plays a dual role in the regulation of telomere length. It shortens excessively long telomeres and elongates short telomeres simultaneously in one cell, maintaining the optimal telomere length at each chromosomal end for efficient protection. This novel hTERT-mediated telomere-shortening mechanism not only exists in cancer cells, but also in primary human cells. The hTERT-mediated telomere shortening requires hTERT’s enzymatic activity, but the telomerase RNA component, hTR, is not involved in that process. We found that expression of hTERT increases telomeric circular DNA formation, suggesting that telomere homologous recombination is involved in the telomere-shortening process. We further demonstrated that shelterin protein TPP1 interacts with hTERT and recruits hTERT onto the telomeres, suggesting that TPP1 might be involved in regulation of telomere shortening. This study reveals a novel function of hTERT in telomere length regulation and adds a new element to the current molecular model of telomere length maintenance.     

端粒及端粒酶的研究进展

端粒是染色体末端独特的蛋白质-DNA结构,在保护染色体的完整性和维持细胞的复制能力方面起着重要的作用.端粒酶则是由RNA和蛋白质亚基组成的、能够延长端粒的一种特殊反转录酶.端粒长度和端粒酶活性的变化与细胞衰老和癌变密切相关.端粒结合蛋白可能通过调节端粒酶的活性来调节端粒长度,进而控制细胞的衰老、永生化和癌变.研制端粒酶的专一性抑制剂在肿瘤治疗方面有着广阔的前景.