端粒,端粒酶及其生物学功能的研究进展

真核生物染色体末端复制,ＤＮＡ聚合酶并不能完成,需要端粒酶来进行,在缺少端粒酶活性的情况下,细胞将发生衰老并直至死亡。在肿瘤细胞中,通过抑制端粒酶活性可达到治疗癌症的目的。构建具有端粒酶活性的反转录酶区表达载体,转化体细胞可获得永生细胞系,可以用于基因治疗和遗传学应用。     

溴化乙锭—TRAP法检测端粒酶活性

端粒酶是一种核蛋白，在真核生物体内，通过将端粒重复顺序转移到染色体末端面维持染色体的稳定。端粒酶活性存在于生殖细胞和永生癌细胞，但在正常细胞中却极低乃至无法检测，我们使用ＥＢ染色简化了端粒酶活性检测的传统的ＴＲＡＰ法。     

溴化乙锭-TRAP法检测端粒酶活性(英文)

端粒酶是一种核蛋白,在真核生物体内,通过将端粒重复顺序转移到染色体末端而维持染色体的稳定．端粒酶活性存在于生殖细胞和永生癌细胞,但在正常细胞中却极低乃至无法检测．我们使用ＥＢ染色简化了端粒酶活性检测的传统的ＴＲＡＰ（Ｔｅｌｏｍ ｅｒｉｃ Ｒｅｐｅａｔ Ａｍ ｐｌｉｆｉｃａｔｉｏｎ Ｐｒｏｔｏｃｏｌ）法．结果表明,对粗抽提物而言,ＥＢ染色具有足够的可信度和灵敏度,同时又经济省时,便于推广使用     

三氧化二砷对肝癌BEL-7402细胞生长及端粒酶活性的影响

目的：观察不同浓度三氧化二砷作用不同时间对肝癌BEL—7402细胞生长及端粒酶活性的影响并探讨其作用机理。方法：应用不同浓度三氧化二砷作用于肝癌细胞系BEL—7402不同时间后，观察肝癌细胞的存活；用端粒酶检测试剂盒检测端粒酶活性的变化。结果：三氧化二砷可显抑制肝癌细胞系BEL—7402的生长；经三氧化二砷作用后肝癌细胞端粒酶活性下降；抑制作用与时间、剂量有关。结论：三氧化二砷可明显抑制肝癌细胞系BEL—7402的生长，其机理可能是降低细胞端粒酶活性和其他的机制。     

关于端粒酶研究:结果与问题

端粒酶是一种由RNA和蛋白质构成的复合结构，在活性状态下端粒酶可以其自身的RNA中的内设模板区为模板，以逆转录方式为染色体末端“加尾”。端粒酶活性的恢复是动物克隆成功的关键因素之一。但是，端粒酶活性恢复机制、端粒酶基因表达调控信号及端粒酶活性与衰老体细胞中染色体端粒恢复的关系等问题还有待研究解决。     

三氧化二砷抑制肝癌细胞端粒酶活性的实验研究

目的：观察三氧化二砷对肝癌细胞株BEL-7402和SMMC-7721端粒酶活性的影响。方法：应用端粒酶多聚酶联反应一酶联免疫测定(PCR-ELISA)方法，检测三氧化二砷作用后，肝癌细胞系BEL-7402和SMMC-7721细胞端粒酶活性的变化并比较两种细胞端粒酶对三氧化二砷敏感性的差异。结果：0．25～2．00μmol／L三氧化二砷(24～96h)可抑制肝癌细胞系BFL-7402的端粒酶活性，抑制作用与时间、剂量呈依赖性关系；0．25～0．50μmol／L三氧化二砷对SMMC-7721细胞端粒酶活性没有影响(96h以内)，1．00～2．00μmol/L三氧化二砷可抑制SMMC-7721细胞端粒酶活性(4H8～96h)，有时间、剂量依赖关系。结论：三氧化二砷可以抑制肝癌细胞系BEL-7402和SMMC-7721细胞端粒酶的活性；BEL-7402细胞端粒酶对三氧化二砷的敏感性较SMMC-7721细胞端粒酶高。     

端粒酶与妇科肿瘤诊治的研究进展

端粒酶是一种RNA依赖的逆转录酶,其激活是细胞永生化和肿瘤形成的关键步骤。在妇科肿瘤的发生过程中,端粒酶活性和hTERT的表达与肿瘤的进展具有相关性。端粒酶及其催化亚单位成为妇科肿瘤早期诊断和预后判断的生物学指标。因此,抑制端粒酶活性有望成为妇科肿瘤治疗的新策略。     

胃肠道恶性肿瘤的端粒酶活性研究

目的：探讨端粒酶活性与胃肠道恶性肿瘤之间的关系．方法：采用非核素银染TRAP法，对30例胃肠道恶性肿瘤(16例胃癌、9例食管癌、5例结肠癌)患者癌组织及其癌旁组织和6例非肿瘤患者的相应正常组织中端粒酶活性进行检测．结果：端粒酶活性在胃癌组织中阳性率为87．5％(14／16)、食管癌中为88．9％(8／9)、结肠癌中为80％(4／5)，其阳性率与患者年龄及组织类型无关，病灶越大、病期越晚、分化程度越低，端粒酶活性率越高，淋巴结转移者高于无转移者．在相应癌旁组织和非肿瘤患者正常组织端粒酶活性皆为阴性．结论：端粒酶活性可能是特异性较强的恶性肿瘤标志物，它有望成为有力的胃肠道恶性肿瘤鉴别诊断的辅助手段，并在肿瘤患者的预后判断方面具重要意义．     

农杆菌介导的红豆草转化及其特性

胭脂碱型根癌农杆菌C58和T37菌株通过离体感染红豆草下胚轴和子叶,能形成激素自主性转化瘤组织、畸胎瘤、再生苗和再生完整植株.在再生苗和植株基部可形成次生瘤组织.对转化瘤组织细胞系与正常愈伤组织细胞系的生理生化分析表明,两者可溶性蛋白质含量没有明显差异,但转化系过氧化物酶活性和L-苯丙氨酸解氨酶活性却比正常愈伤组织细胞系高得多.这可能与转化瘤组织中致瘤基因onc的表达改变内源激素水平所致.游离氨基酸含量的分析表明,转化瘤组织的总游离氨基酸含量比正常愈伤组织细胞系高2-3倍,其中除转化瘤组织中的天门冬氨酸含量比正常愈伤组织细胞系高18-37倍,C58转化瘤组织和T37转化瘤组织也显示出明显差异,其中,T37转瘤组织中的谷氨酸,精氨酸和脯氨酸含量比C58转化瘤组织和正常愈伤组织细胞系高得多的.     

MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans

The germ line is an immortal cell lineage that is passed indefinitely from one generation to the next. To identify the genes that are required for germline immortality, we isolated Caenorhabditis elegans mutants with mortal germ lines--worms that can reproduce for several healthy generations but eventually become sterile. One of these mortal germline (mrt) mutants, mrt-2, exhibits progressive telomere shortening and accumulates end-to-end chromosome fusions in later generations, indicating that the MRT-2 protein is required for telomere replication. In addition, the germ line of mrt-2 is hypersensitive to X-rays and to transposon activity. Therefore, mrt-2 has defects in responding both to damaged DNA and to normal double-strand breaks present at telomeres. mrt-2 encodes a homologue of a checkpoint gene that is required to sense DNA damage in yeast. These results indicate that telomeres may be identified as a type of DNA damage and then repaired by the telomere-replication enzyme telomerase.     

Comparative pluripotency analysis of mouse embryonic stem cells derived from wild-type and infertile hermaphrodite somatic cell nuclear transfer blastocysts

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer （SCNT）, holds great promise for treating many human diseases using regenerative medicine. Teratoma formation and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells （hESCs） have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmis- sion, which would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines lack full multi- or pluripotency has not yet been evaluated. In this study, we established 16 mouse ESC lines, including 3 genetically defective nuclear transfer- ESC （ntESC） lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted via the germ line, in contrast with normal ntESCs. Our results in- dicate that teratoma formation assays with hESCs might be an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards are required to assess the safety of hESCs for therapeutic cloning.     

Evolution of ageing

An evolutionary view of ageing suggests that mortality may be due to an energy-saving strategy of reduced error regulation in somatic cells. This supports Orgel's 'error catastrophe' hypothesis and offers a new basis for the study of normal and abnormal ageing syndromes and of apparently immortal transformed cell lines.     

GDNF提高端粒酶活性并且促进神经干细胞生长和分裂

对于多种神经细胞，胶质细胞源性神经营养因子（GDNF）具有维持生长、存活、促进分化和成熟的作用，初次在神经干细胞中发现了GDNF的这一营养效应，但这种作用并不对神经干细胞的分化方向产生影响，进而通过测定神经干细胞的端粒酶活性和端粒酶活性的抑制实验，表明端粒酶活性的增高与GDNF对神经干细胞生长和分裂的促进作用有关，因此完善了GDNF在神经细胞中的信号传导和功能实现途径的理解。     

Generation of pluripotent stem cells from adult human testis

Human primordial germ cells and mouse neonatal and adult germline stem cells are pluripotent and show similar properties to embryonic stem cells. Here we report the successful establishment of human adult germline stem cells derived from spermatogonial cells of adult human testis. Cellular and molecular characterization of these cells revealed many similarities to human embryonic stem cells, and the germline stem cells produced teratomas after transplantation into immunodeficient mice. The human adult germline stem cells differentiated into various types of somatic cells of all three germ layers when grown under conditions used to induce the differentiation of human embryonic stem cells. We conclude that the generation of human adult germline stem cells from testicular biopsies may provide simple and non-controversial access to individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells.     

A telomerase component is defective in the human disease dyskeratosis congenita

The X-linked form of the human disease dyskeratosis congenita (DKC) is caused by mutations in the gene encoding dyskerin. Sufferers have defects in highly regenerative tissues such as skin and bone marrow, chromosome instability and a predisposition to develop certain types of malignancy. Dyskerin is a putative pseudouridine synthase, and it has been suggested that DKC may be caused by a defect in ribosomal RNA processing. Here we show that dyskerin is associated not only with H/ACA small nucleolar RNAs, but also with human telomerase RNA, which contains an H/ACA RNA motif. Telomerase adds simple sequence repeats to chromosome ends using an internal region of its RNA as a template, and is required for the indefinite proliferation of primary human cells. We find that primary fibroblasts and lymphoblasts from DKC-affected males are not detectably deficient in conventional H/ACA small nucleolar RNA accumulation or function; however, DKC cells have a lower level of telomerase RNA, produce lower levels of telomerase activity and have shorter telomeres than matched normal cells. The pathology of DKC is consistent with compromised telomerase function leading to a defect in telomere maintenance, which may limit the proliferative capacity of human somatic cells in epithelia and blood.     

端粒酶核酶抑制肿瘤细胞生长的研究

已证明有84.7%的人类恶性肿瘤组织细胞中端粒酶表达异常增高.端粒酶的作用是合成染色体末端端粒重复序列维持染色体稳定及细胞永生化的关键因素.因此,端粒酶在肿瘤发生发展中起重要作用,是目前最广泛的肿瘤标志物.核酶是一种具有内切酶活性的RNA分子,只要满足一定的空间结构就能定点切割RNA底物.切割端粒酶的核酶(简称为端粒酶核酶)主要针对端粒酶的两个亚单位hTR和hTERT抑制两基因的表达,降低端粒酶的活性,抑制肿瘤细胞的生长.     

端粒酶转染防治角膜内皮失代偿的研究

目的评价端粒酶转染对角膜内皮细胞形态和功能的影响。方法将体外培养的猫角膜内皮细胞随机分为3组,兔端粒酶基因转染组、正常对照组和表皮生长因子组。培养30代后观察传代细胞形态和细胞周期各时相细胞比例的变化,检测端粒酶逆转录酶蛋白和Ⅳ型胶原的表达。结果兔端粒酶基因转染组细胞数目较正常对照组和表皮生长因子组增多,伸展贴壁能力增强,表型正常,G2～M期和S期细胞比例显著增加,端粒酶逆转录酶蛋白和Ⅳ型胶原的表达也较正常对照组增多。结论兔端粒酶转染可以促进猫角膜内皮细胞增殖且表型正常,可以增强其伸展粘附能力和分泌功能,有利于防治角膜内皮失代偿。