肿瘤组织中端粒酶活性的研究

目的 了解端粒酶活性高低与肿瘤恶性程度关系。方法 用ＴＲＡＰ－ＥＬＩＳＡ和我们改进的ＴＲＡＰ银染,对肿瘤组织酶活笥进行半定量检测。结果 端粒酶阳性率分别为：胶质瘤星形Ⅱ级４０％,Ⅲ和Ⅳ级１００％,肠癌８４．６２％,食管癌９０％。结论 端粒酶活性与肿瘤恶性程度密切相关,是诊断恶性肿瘤、判断愈后的良好指标。     

卵巢肿瘤组织中端粒酶逆转录酶的原位表达

目的　探讨卵巢肿瘤组织中端粒酶逆转录酶 (TRT)的表达及其与凋亡基因 p5 3蛋白和bcl 2基因的关系。方法　应用免疫组织化学S P法检测 78例卵巢肿瘤组织中TRT及p5 3、bcl 2的表达 ,结果进行统计学分析。结果　TRT在不同卵巢肿瘤组中的表达差异有显著性 ,在卵巢上皮组织肿瘤中良性、交界性和恶性肿瘤组的阳性表达分别为 6 7%、5 7 2 %和 85 % ,在卵巢生殖细胞肿瘤中良性与恶性的阳性表达为 10 %和 81 8% ,TRT的表达随着肿瘤恶性度增高呈现阳性率和阳性强度递增现象 ,两者比较差异有显著性 (P <0 0 1) ;卵巢恶性上皮组织肿瘤中 p5 3蛋白和bcl 2基因的阳性表达率分别为 90 %和80 % ,相关分析显示TRT与 p5 3和bcl 2的表达密切相关 (P <0 0 1)。结论　TRT的高表达与卵巢恶性肿瘤有密切关系 ,卵巢肿瘤组织中TRT与p5 3、bcl 2的表达呈正相关 ,TRT可作为卵巢肿瘤恶性度的一个指标。     

鼻咽上皮癌变过程中端粒酶活性和端粒酶RNA的表达

目的 研究二亚硝基哌嗪（DNP）诱发大鼠鼻咽癌变过程中端料酶的表达规律。方法 以DNP诱导大鼠鼻咽癌：用PCR-ELISA和Nested RT-PCR检测DNP诱导大鼠鼻咽癌变不同阶段端粒酶活性和端料酶RNA的表达,同时作病理形态学检测。结果 DNP诱导大鼠鼻咽癌变过程中,端粒酶活性不断升高,端粒酶的变化与鼻咽癌变呈正相关,而且端粒酶中RNA表达先于端粒酶的表达。在大鼠鼻咽上皮细胞异型增生阶段即出现端粒酶的激活和端粒酶RNA的表达。结论 化学致癌物DNP诱导细胞癌变端粒酶的激活,且端粒酶的扩活和端粒酶RNA的表达是鼻咽上皮癌变的早发事件,与鼻咽癌的发生、发展有关。     

大肠癌中端粒酶RNA的表达及意义

目的：探讨端粒酶RNA（hTR)表达在大肠癌发生、发展的意义以及与细胞增殖的关系。方法：应用原位杂交方法检测26例大肠癌、18例大肠腺癌、18例大肠息肉及5例炎症病变中端粒酶hTR的表达。应用免疫组化En Vision法检测Ki-67抗原的表达,反映细胞增殖活性。结果：26例大肠癌tTR以强阳性表达为主（21／26,80．8％）;18例大肠腺癌hTR大部分呈弱阳性表达（12／18,66．7％）,少数呈强阳性（5／18,27．8％）;息肉、炎症hTR为弱表达。端粒酶hTR表达在大肠良、恶性病变中存在差异（P＜0．01）,并且随肿瘤分化程度的下降、浸润深度的增加以及淋巴结转移的发生,hTR表达增强（P＜0．05）。此外,在细胞增殖活跃区域,显示出高水平hTR的表达。结论：在大肠肿瘤发生早期就有端粒酶hTR表达的上调,而且hTR表达与肿瘤分化程度、浸润和转移等呈正相关。hTR表达也与细胞增殖活性相关。用原位杂交方法检测端粒酶hTR的表达,有助于大肠肿瘤的早期诊断以及生物学行为的评估。     

人非小细胞肺癌细胞中端粒酶活性的检测与研究

目的 研究非小细胞肺癌细胞癌组织中端粒酶活性表达,探讨端粒酶生表达与非小细胞肺癌发生发展的关系。方法 收集经手术及病理证实的非小细胞肺癌４８例标本,１２例肺癌癌旁肺组织、７例非肿瘤病例所取正常肺组织作对照。用端粒酶检测试剂盒检测组织本端粒酶活性。结果 ７５．００％（３６／４８）非小细胞肺癌组织标本检出端粒酶活性,８．３３％（１／１２）癌旁肺组织检出端粒酶活笥,７例非肿瘤标本所取的正常肺组织均未检出     

食管癌及癌旁组织中端粒酶活性检测及其意义

目的探讨食管癌及癌旁组织中端粒酶活性检测的意义. 方法采用 PCR技术为基础的 TRAP法检测了 39例食管癌及癌旁组织端粒酶活性. 结果 39例食管癌组织中 34例端粒酶活性阳性,阳性率为87.2%,癌旁组织中3例阳性,阳性率为7.7%,9例食管良性组织中1例阳性,阳性率为11.1%.食管癌与相应癌旁组织和良性食管组织端粒酶阳性检测率相比,差异有显著性意义(p<0. 01).伴有淋巴结转移的 25例食管癌端粒酶阳性检测率为96.0%,显著高于未伴有淋巴结转移的阳性率(71.4%),两者差异有显著性意义(p<0.05). 结论端粒酶激活与食管癌发生发展有关,对端粒酶活性进行检测对食管癌的诊断和判断预后有重要价值.     

食管癌及癌旁组织中端粒酶活性检测及其意义

目的探讨食管癌及癌旁组织中端粒酶活性检测的意义.方法采用PCR技术为基础的TRAP法检测了39例食管癌及癌旁组织端粒酶活性.结果39例食管癌组织中34例端粒酶活性阳性,阳性率为87.2%,癌旁组织中3例阳性,阳性率为7.7%,9例食管良性组织中1例阳性,阳性率为11.1%.食管癌与相应癌旁组织和良性食管组织端粒酶阳性检测率相比,差异有显著性意义(p<0.01).伴有淋巴结转移的25例食管癌端粒酶阳性检测率为96.0%,显著高于未伴有淋巴结转移的阳性率(71.4%),两者差异有显著性意义(p<0.05).结论端粒酶激活与食管癌发生发展有关,对端粒酶活性进行检测对食管癌的诊断和判断预后有重要价值.     

胃镜活检胃癌组织中端粒酶的定量表达

目的通过检测端粒酶在人胃镜活检胃癌组织中的定量表达情况,然后分析其与术后胃癌标本的临床病理因素之间的关系,从而为临床医生术前预测胃癌的浸润及转移情况及选择合理的治疗方式提供一定的理论依据。方法应用Trizol法提取30例胃镜活检胃癌组织中总的RNA,以总RNA为模板经逆转录合成cDNA,再用实时荧光定量PCR的方法检测该30例胃镜活检胃癌组织中端粒酶的表达情况并分析其与各种临床病理指标的关系。结果荧光实时定量PCR扩增结果显示,胃癌组织中端粒酶的表达与浸润深度、分化程度及淋巴结转移密切相关(P0.05)。而且端粒酶在黏液腺癌中的表达远远高于在腺癌中的表达(P0.05)。结论通过该实验证明端粒酶在胃镜活检胃癌组织中的表达与胃癌侵袭转移密切相关,有可能成为术前预测胃癌侵袭转移的重要预后指标,进而为临床医生选择治疗方案及手术方式提供一定的临床参考价值。     

骨巨细胞瘤组织中端粒酶活性的原位检测及意义

目的:探讨骨巨细胞瘤(GCT)石蜡切片中端粒酶活性检测的可行性以及端粒酶在GCT生物学特性中的意义。方法:采用针对人端粒酶hTERT寡核苷酸探针序列, 对27例GCT石蜡标本进行原位杂交, 检测端粒酶逆转录酶(hTERT)mRNA的表达情况。结果:27例GCT中8例hTERTmRNA表达阳性, 石蜡切片中基质细胞、部分多核巨细胞hTERTmRNA表达阳性。结论:采用原位杂交技术检测GCT石蜡切片中hTERT表达, 证实GCT中可能存在肿瘤性巨细胞。     

结直肠癌组织中端粒酶和pRb表达及其临床意义

目的：观察端粒酶和视网膜母细胞瘤基因蛋白（pRb）在结直肠癌中的表达,并探讨它们之间的相关性。方法：应用免疫组化法检测48例结直肠癌组织中端粒酶和pRb的表达,并用统计学方法分析表达结果与结直肠癌临床病理因素间的关系。结果：结直肠癌中端粒酶和pRb的阳性表达率分别为87．5％和56．3％,且与结直肠癌区域淋巴结转移和临床分期关系密切（P〈0．05）,端粒酶表达还与组织病理学分级密切相关（P〈0．05）。结论：检测结直肠癌组织中端粒酶和pRb的表达有助于结直肠癌患者的预后评估,结直肠癌组织中端粒酶与pRb表达呈显著负相关关系,两者可能从不同方面共同促进结直肠癌的发生。     

原发性胃癌组织中的端粒及端粒酶表达

目的观察端粒（ＴＲＦ）及端粒酶在胃癌形成及发展中的作用。方法采用ＤＮＡ琼脂糖凝胶杂交技术及端粒重复扩增ＰＣＲ方法检测１７例早期胃癌、８９例进展期胃癌组织及邻近正常组织中的平均ＴＲＦ长度及端粒酶活性。结果早期、进展期胃癌的ＴＲＦ长度及端粒酶活性表达均显著短于或高于邻近胃组织（Ｐ＜０．０５～０．０１），且端粒酶活性的表达主要表现在ＴＲＦ缩短或延长的胃癌组织中，而ＴＲＦ缩短与胃癌的分化程度相一致。结论端粒及端粒酶的异常状态可能与胃癌的发生发展密切相关，端粒酶活性及端粒缩短可以作为胃癌的诊断标志     

端粒酶转录酶及其调节相关蛋白与增殖细胞核抗原在卵巢上皮性肿瘤中的表达及临床意义

目的　探讨端粒酶转录酶 (hTERT)及端粒酶调节相关蛋白 (TRAP)与增殖细胞核抗原(PCNA)在卵巢上皮性肿瘤中的表达和临床意义。方法　收集 10 6例卵巢上皮性肿瘤 (恶性 5 4例 ,交界性 33例 ,良性 19例 )的临床资料 ,行hTERT、TRAP和PCNA 3种抗体的免疫组织化学标记链霉素卵白素生物素 (LSAB)法染色。对 87例恶性和交界性患者进行了随访 ,4 5例获结果 ,随访时间为 2～6 0个月。结果　hTERT蛋白的表达在良性 (4/ 19)和交界性 (90 9% ,30 / 33)以及良性和恶性(94 4 % ,5 1/ 5 4 )间的差异均有显著性 (P均 <0 0 0 1) ,TRAP蛋白的表达在良性 (4/ 15 )和恶性(77 8% ,2 8/ 36 )间的差异有显著性 (P <0 0 0 1) ;hTERT和TRAP蛋白的表达在卵巢癌Ⅰ、Ⅱ期和Ⅲ、Ⅳ期两组病例中的差异均无显著性 (P >0 0 5 ,P >0 3)。PCNA的表达在良性 (6 9± 5 9) %和交界性 (2 6 4± 17 8) %、良性和恶性 (5 1 8± 2 2 1) %以及交界性和恶性间的差异均有显著性 (P <0 0 1,P <0 0 0 1,P <0 0 5 )。 33例交界性患者全部存活 ,5 4例恶性患者中 35例 (6 4 8% )有转移 (包括 5例淋巴结转移 ) ,4例 (7 4 % )死亡。结论　hTERT和TRAP蛋白的表达与卵巢上皮性肿瘤的良恶性有关 ,但与其临床分期无关 ;hTERT和TRAP蛋白的表达相似     

端粒酶活性联合CA125检测与预测卵巢上皮癌腹腔化疗疗效的关系

目的：对卵巢上皮癌术前或每次化疗前血清癌抗原125（cancer antigen 125,CA125）与腹水或腹腔冲洗液中脱落细胞端粒酶活性的检测结合，以供评价腹腔化疗疗效。方法：收集35例术前或每次化疗前血清，采用ELISA法测定血清CA125；并且收集其腹水或腹腔冲洗液中脱落细胞，用TRAP-PCR-ELISA银染法检测腹水脱落细胞端粒酶活性。结果：(1) 卵巢上皮癌腹水或腹腔冲洗液中85.7%为端粒酶阳性;而且，端粒酶活性检测在术前为1.54 U±0.82 U，术后第1疗程化疗后为0.79 U±0.69 U,术后第2疗程化疗后为0.55 U±0.80 U,术后第3疗程化疗后为0.46 U±0.71 U (P＜0.05),差异显著。（2）根据患者的临床化疗效应，得出血清CA125表达水平 ≥50% 或＜50%时，卵巢上皮癌化疗敏感和耐药的准确性分别为91%和69%;并且端粒酶活性表达水平下降或升高时，卵巢上皮癌临床化疗敏感和耐药的准确性分别为88%和80%；如果将其CA125与端粒酶活性表达水平下调结合判定，得出对化疗敏感或耐药的预估值达95%和100%的提高化疗疗效结果。结论：端粒酶活性检测可能较准确地预测卵巢上皮癌临床腹腔化疗疗效，它与血清CA125联合有利于提高临床化疗疗效的判定。     

Telomere length and telomerase activity: effect of ageing on human NK cells

Telomeres are repeats of TTAGGG sequences located at the end of eukaryotic chromosomes. They are essential for stabilisation and protection of chromosomal ends and for the regulation of cell replicative capacity. Due to the end-replication defect of DNA polymerase, telomeres shorten progressively with each cell division and telomere length may be an indicator of the replicative history of a cell. Compensatory mechanisms for the telomere loss have been identified. The most widely studied one is mediated by telomerase a ribonuclear protein-enzyme complex that synthesise telomeric repeats. In this study we have investigated whether NK cells, derived from a group of old healthy subjects, underwent the modifications of telomere length and telomerase activity observed in other sub-populations of lymphocytes with advancing age. We demonstrated that: (a) telomere shortening occurred and telomerase activity decreased in human NK cells with ageing; (b) the rate of telomere loss was different under and over 80 years of age; (c) similarly to telomere shortening, the modification of telomerase activity was particularly evident in octogenarians; (d) subjects with the most evident modifications of telomeres and telomerase were the oldest and those with increased NK cell numbers.     

The teleost Oryzias latipes shows telomere shortening with age despite considerable telomerase activity throughout life

Previous studies of telomeres and telomerase have focused mostly on mammals, and data for other vertebrates are limited. We analyzed both telomere length (terminal restriction fragment length) and telomerase activity in a small freshwater teleost fish, the medaka (Oryzias latipes), and found that the telomeres shorten during ageing despite the fact that a considerable amount of telomerase activity is ubiquitously detectable throughout the life of the fish. Since the telomere attrition rate during development was greater than that in adulthood, telomere length is inversely correlated with the increase in body length. The difference in telomere length among medaka individuals was similar to that in humans, and the individual specific differences were evident even at the earliest embryonic stage. Telomerase activity was ubiquitously detectable not only in the body of the embryo but also in the systemic organs of mature individuals throughout their entire life span. These data suggest that telomere attrition during ageing in medaka, which is similar to that in humans, may be a major factor determining their mortality, and that telomere maintenance through strong telomerase activity may be required for the characteristic lifelong continuous growth of this fish.     

白血病细胞中端粒酶抑制因子Pinx1的表达与端粒酶活性关系的研究

目的：研究端粒酶抑制因子Pinx1在急性白血病细胞中的表达和在急性早幼粒白血病细胞株细胞NB4分化过程中的表达改变，分析其表达与端粒酶逆转录酶hTERT表达的关系，以了解白血病细胞中Pinx1对端粒酶的作用及可能机制。方法:荧光定量RT-PCR检测30例急性白血病细胞中Pinx1与hTERT mRNA的表达，进一步在ATRA诱导的急性早幼粒白血病细胞株细胞NB4分化过程中检测Pinx1及hTERT mRNA表达，分析两者之间的相关性。结果:Pinx1在急性白血病细胞中的表达（0.00312，5.42×10-4-0.024）明显高于正常人骨髓单个核细胞（7.89×10-4，0-0.00863，P＜0.01），与hTERT的表达呈正相关（r=0.296,P＜0.05）。Pinx1表达随NB4细胞分化逐渐下降，与hTERT呈正相关（r=0.900,P＜0.05） 。结论：Pinx1虽然为端粒酶活性的抑制因子，但在白血病细胞中与端粒酶活性的调控方向一致，提示Pinx1的表达改变可能是继发于hTERT的一种负反馈反应，目的是保持端粒酶活性的稳定，具体机制尚待进一步研究。     

端粒/端粒酶在恶性血液病中的研究进展

端粒是真核细胞染色体末端特有的一段DNA序列和几种特异性蛋白质构成的复合物。端粒酶是一种核糖核酶,能以自身RNA为模板,合成端粒DNA,从而维持端粒的长度。端粒/端粒酶的表达调控对肿瘤的发生发展及细胞的衰老、永生化起着重要的作用,深入研究端粒/端粒酶的结构功能及其在恶性血液病细胞和正常造血细胞中的表达和调控,将有助于达到控制和治疗恶性血液病的目的。     

Massive telomere loss and telomerase RNA expression in dexamethasone-induced apoptosis in mouse thymocytes

Apoptosis is a well-recognized process of cell death occurring under several physiological and pathological conditions and represents the principal mechanism involved in cell selection in the thymus. Glucocorticoids are well known to stimulate apoptosis in rat thymocytes. However, it is unclear whether the same changes occur after in vivo glucocorticoid treatment in mice. Chromosomal stability and cell viability require a proficient telomeric end-capping function. Cells with critical telomere shortening and telomerase dysfunction undergo increased apoptosis. In turn, the change in telomere function in cells undergoing apoptosis is not fully characterized. In order to investigate this, we studied the changes in thymocytes after dexamethasone administration in BALB/c mice. The loss of normal thymocytes coincided with the appearance of small dense cells with characteristic features of apoptosis including condensed chromatin, internucleosomal DNA cleavage, and a "hypodiploid" peak on flow cytometry, which suggested that dexamethasone-induced thymocyte apoptosis in BALB/c mice could be considered a well-defined experimental model for studying apoptotic processes. Dexamethasone-treated thymocytes exhibited rapid and dynamic loss of telomeric sequences and up-regulation of telomerase RNA as an early event in the apoptotic process. Telomerase activity was unchanged in this event. Thereafter, telomere gain associated with an increase in telomerase activity occurred in the regenerative process of the thymus. These results suggest a role of telomere loss and up-regulation of telomerase RNA as key apoptosis sensors.     

Telomere length and telomerase activity during expansion and differentiation of human mesenchymal stem cells and chondrocytes

Chondrocyte ex vivo expansion currently performed to replace damaged articular surfaces is associated with a loss of telomeric repeats similar to decades of aging in vivo. This might affect the incidence or time of onset of age-related disorders within transplanted cells or tissues. This study examined whether more immature progenitor cells, such as mesenchymal stem cells (MSC), which can be expanded and subsequently differentiated into chondrocytes is advantageous regarding telomere-length related limitations of expansion protocols. Primary chondrocytes and bone-marrow-derived MSC were isolated from 12 donors, expanded separately to 4 x 10(6) cells, and (re-)differentiated as three-dimensional chondrogenic spheroids. Cells were collected during expansion, after three-dimensional culturing and chondrogenic differentiation, and sequential analyses of telomere length and telomerase activity were performed. Surprisingly, telomeres of expanded MSC were significantly shorter than those from expanded chondrocytes from the same donor (11.4+/-2.5 vs. 13.4+/-2.2 kb) and tended to remain shorter after differentiation in chondrogenic spheroids (11.9+/-1.8 vs. 13.0+/- kb). While telomere lengths in native chondrocytes and MSC were not related to the age of the donor, significant negative correlations with age were observed in expanded (136 bp/year), three-dimensionally reconstituted (188 bp/year), and redifferentiated (229 bp/year) chondrocytes. Low levels of telomerase activity were found in MSC and chondrocytes during expansion and after (re-)differentiation to chondrogenic spheroids. In terms of replicative potential, as determined by telomere length, ex vivo expansion followed by chondrogenic differentiation of MSC did not provide a benefit compared to the expansion of adult chondrocytes. However, accelerated telomere shortening with age during expansion and redifferentiation argues for an "age phenotype" in chondrocytes as opposed to MSC and suggests an advantage for the use of MSC especially in older individuals and protocols requiring extensive expansion