乳癌组织端粒酶活性表达及其与p16基因的关系

目的：探讨乳房恶性肿瘤组织中端粒酶活性的表达情况及ｐ１６抑癌基因的影响。方法：应用端粒重复放大程序－酶标法（ＴＲＡＰ－ＥＬＩＳＡ）及ＴＲＡＰ－银染法检测３５例有乳癌组织及２４例良性肿瘤组织中的端粒酶活性,并采用多重ＰＣＲ技术对乳癌组织的ｐ１６基因的缺失空谈进行了分析,结果：３５例乳癌标本中有２８例端粒酶活性表达为阳性（８０％）,而２４例乳房良性肿瘤标本中仅１例端粒酶活性表达阳性（４．１％）。此外３５例乳癌标本中的１５例存在有ｐ１６基因的外显子２及外显子１的缺失（４２．８％）,而该１５例中有１４例被检测为端粒酶活性阳性（９３．３％）,但２０例未缺失标本的端粒酶阳怀率仅为７０％（１４／２０）。结论：端粒酶活性与乳房肿瘤组织的恶性程度密切相关,提示端粒酶参与了肿瘤的形成过程,而端粒酶活性的增高与ｐ１６抑癌基因的缺失突     

原发性肝癌及慢性肝脏病变端粒酶活性的研究

目的比较原发性肝细胞癌（ＨＣＣ）及其癌旁不同慢性病变组织端粒酶活性的异同,探讨端粒酶活性在恶性肿瘤诊断中的意义。方法用ＴＲＡＰ方法检测３８例ＨＣＣ及其癌旁不同慢性病变肝组织的端粒酶活性。同时以４例正常肝组织为对照。结果３８例ＨＣＣ中３２例显示端粒酶活性（８６８％）。端粒酶活性与ＨＣＣ的细胞分化、类型、大小及有无乙型肝炎病毒（ＨＢＶ）感染无关,但与病人血清中甲胎蛋白（ＡＦＰ）水平相关。端粒酶活性阴性组ＡＦＰ水平明显低于各阳性组（Ｐ＜００１）。正常肝（４例）、先天性胆管闭锁（４例）、肝小叶间纤维增生（２例）、慢性病毒性肝炎（２例）及未见明显病变肝组织（７例）均未见有端粒酶活性。２５例肝硬化中４例端粒酶呈弱活性。结论端粒酶活性见于大多数ＨＣＣ及个别肝硬化组织,其活性可能在ＨＣＣ的发生、发展中起重要作用,有希望成为恶性肿瘤诊断的标记物。     

肝细胞癌及癌旁肝组织中bcl—2和p53蛋白表达与？…

目的：探讨ｂｃｌ－２和ｐ５３蛋白的表达与端粒酶活性的相关性及其与ＨＣＣ发生的关系。方法：利用端粒酶原位标记法显示端粒酶活性,采用Ｓ－Ｐ法免疫组化技术的检测ｂｃｌ－２和ｐ５３蛋白。结果：端粒酶在ＨＣＣ中的阳性率（９１．７％）显著高于癌旁肝组织（５８．３％）（Ｐ〈０．０５）,端粒酶活性强度与ＨＣＣ分化程度无关（Ｐ〉０．０５）;癌组织中ｂｃｌ－２和ｐ５３蛋白的阳性率均高于癌旁组织（Ｐ〈０．０１）;ＨＣＣ     

星形细胞肿瘤中端粒酶与PTEN的表达及相关性

目的：研究星形细胞肿瘤中的端粒酶活性和PTEN表达,探讨其在星形细胞肿瘤发生发展过程中的相关性,方法：收集手术切除经病理证实的星形细胞肿瘤110例,8例正常脑组织作对照。用端粒酶原位杂交检测盒检测组织标本端粒酶活性,用LSAB法检测PTEN表达。结果：星形细胞肿瘤端粒酶活性和PTEN的阳性检出率分别为50％（55／110）和39．1％（43／110）,且随着肿瘤恶性程度的增加,端粒酶活性程度升高（P＜0．01）,端粒酶活性与PTEN表达有相关性（P＜0．01）,结论：端粒酶活性与星形细胞肿瘤的分化程度有关,提示端粒酶活性可能是星形细胞肿瘤恶性程度的重要标记物,PTEN蛋白可能对端粒酶的活性有调节作用。     

乳腺良恶性病变组织端粒长度和端粒酶活性检测

目的　比较乳腺良恶性病变端粒长度改变及其在肿瘤发生发展中的意义 ;探讨端粒酶活性与临床病理参数的关系及其在乳腺癌诊断中的价值。方法　Southern印迹杂交检测TRF长度 ,端粒重复扩增分析 (TRAP)方法检测端粒酶活性。结果　乳腺癌组织平均TRF为 (5 2± 2 8)kb ,与正常组织比较明显缩短 (P <0 0 0 1) ,从正常乳腺组织到乳腺良性病变、乳腺原位癌及浸润性癌平均TRF呈递减趋势。 5 8例乳腺癌中 4 9例端粒酶阳性 (84 7% ) ,端粒酶活性与临床病理参数无相关性 ;癌旁组织端粒酶为阴性 ,而 7例乳腺增生症和 6例乳腺纤维腺瘤中分别有 1例端粒酶阳性 ,与乳腺癌比其差异有显著性 (P <0 0 0 1)。结论　端粒长度在肿瘤发生发展过程中渐进性缩短 ,并最终触发端粒酶的激活 ;端粒酶活性检测有望成为乳腺癌诊断的可靠标记物     

端粒酶检测方法的新进展

端粒酶是催化合成并维持端粒一定序列的一种核糖核蛋白，是一种专一的逆转录酶。目前端粒酶的检测方法包括：（１）基本方法；（２）ＴＲＡＰ法；（３）ＥＬＩＳＡ法；（４）接近闪烁分析法；（５）原位杂交法。大多数肿瘤细胞都有端粒酶活性表达，端粒酶与肿瘤细胞存在高度相关性，端粒酶检测方法的不断发展为肿瘤的诊断和治疗提供了新途径     

端锚酶和端粒酶反义寡核苷酸联合作用对人肺腺癌细胞 A549 端粒的影响

目的探讨端锚酶及端粒酶反义寡核苷酸联合作用对A549细胞端粒相关蛋白表达和翻译及对端粒缩短和细胞周期的作用。方法将A549细胞随机分组为空白对照、sTANKS、shTERT、asTANKS、ashTERT和asTANKS ashTERT组,经不同处理,检测hTERTmRNA、端粒酶及端锚酶活性、端粒平均长度及A549细胞寿命。结果ashTERT能抑制hTERTmRNA表达及蛋白质活性;asTANKS不影响hTERTmRNA表达,却能抑制端锚酶活性。asTANKS或ashTERT均可致A549端粒长度缩短,asTANKS ashTERT则缩短更为明显,其减少A549细胞平均传代数的作用也明显大于asTANKS或ashTERT。结论asTANKS对A549端粒长度的抑制有别于端粒酶途径,不仅能通过影响端锚酶活性缩短肿瘤细胞A549端粒的平均长度,而且可与端粒酶抑制剂协同作用明显缩短肿瘤细胞的生存周期,这可能成为抗癌作用的新靶点。     

卵巢肿瘤组织中端粒酶活性的表达

目的分析端粒酶活性在卵巢肿瘤中的表达,探讨端粒酶活性作为卵巢肿瘤诊断肿瘤标记物的意义。方法取冰冻肿瘤组织,采用端粒重复序列扩增法（Telomeric Repeat Amplication Protocol）,结合银染,共测定了27例卵巢癌、5例良性卵巢肿瘤、22例卵巢癌癌旁组织以及12例正常卵巢组织中端粒酶活性,并分析其与组织分级、FIGO分期、病理类型以及有无转移的关系。PCR产物以采用聚丙烯酰胺凝胶电泳分析,以出现特征性的6-bp间隔的特征性阶梯状条带为阳性。出现〉100bp的条带为强阳性,出现〈99bp的条带为中低阳性。结果本实验共对66份标本进行了检测,12例正常卵巢组织中,仅有2例表达端粒酶活性;5例卵巢良性肿瘤中,1例表达端粒酶弱阳性。27例卵巢恶性肿瘤标本中,有23例表达端粒酶活性（其中强阳性16例）;在检测的22例卵巢上皮癌的癌旁组织中,有12例表达端粒酶弱阳性。卵巢恶性肿瘤的端粒酶活性表达阳性率显著高于卵巢良性肿瘤（P=0.0090）和正常卵巢（P=0.0000）。Ⅰ-Ⅱ期患者端粒酶活性阳性率71%,Ⅲ-Ⅳ期患者端粒酶活性阳性率90%（P=0.269）。而强阳性率Ⅲ-Ⅳ期明显高于Ⅰ-Ⅱ期（P=0.009）。在肿瘤类型和组织学分级中,端粒酶阳性表达率均没有显著差异。结论在卵巢恶性肿瘤组织中端粒酶活性有较高的表达率;早期和晚期卵巢肿瘤组织中端粒酶活性阳性率没有明显差异,但晚期卵巢肿瘤组织中的端粒酶活性程度较高;端粒酶活性有可能作为早期诊断卵巢肿瘤的标记物之一,并可以作为卵巢肿瘤判断预后的指标。     

端粒及端粒酶研究的新进展

端粒是染色体末端高度保守的重复核苷酸序列，对染色体具有保护作用，随着细胞分裂的不断进行，端粒逐渐缩短，减少到一定程度，细胞就趋向衰亡，被认为是细胞有丝分裂的“生物钟”。端粒酶是影响端粒长度的主要因素，以其ＲＮＡ为模板，向端粒末端添加（ＴＴＡＧＧＧ）ｎ序列，使端粒延长，从而延长细胞的寿命甚至使其永生。端粒酶的功能区主要在ｈＴＲ的４４－２０３核苷酸区，３ｐ和１０ｐ上存在着编码调整端粒酶的基因，Ｅｓｔｌ可能是端粒末端结合蛋白，是端粒酶的识别位点。正常情况下，此酶活性较低或无活性，但在干细胞、永生型细胞或肿瘤细胞此酶活性较强。端粒酶的活性主要与细胞分裂速度、细胞周期因素及细胞分化程度有关，细胞分化程度低、细胞增生活跃及肿瘤细胞在Ｓ期时活性较高。在恶性肿瘤中端粒酶活性较高，但在良性肿瘤和非肿瘤中活性较低，因而可利用ＴＲＡＰ技术对端粒酶活性进行检测来进行肿瘤诊断及良恶性鉴别。同时可利用端粒酶抑制剂能抑制端粒酶的活性，缩短端粒，使恶化细胞良转，达到治疗肿瘤的作用，而且还可利用检测端粒酶作为治疗效果好坏的指标。     

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     

Lack of correlation between telomere length and telomerase activity and expression in leukemic cells

The expression of three components of telomerase complex (hTR, hTERT, TP1) along with telomerase activity and telomere length in leukemic cells was investigated. Cells were isolated from peripheral blood and/or bone marrow of children with acute lymphoblastic (ALL) and non-lymphoblastic (ANLL) leukemia. Expression of three components of telomerase as well as telomerase activity was found in all leukemic cells. Chemiluminescent detection of terminal restriction fragments (TRF) from DNA isolated from ALL cells showed variable patterns expressing considerable heterogeneity of telomere length. The ALL cells appeared to have both long and short telomere lengths, in contrast to normal peripheral lymphocytes, which produced limited pattern of TRF. The ANLL cells produced predominantly short telomere pattern despite high telomerase activity and expression. It can be concluded that high telomerase activity and expression in leukemic cells is not always correlated with long telomeres (TRF pattern).     

Equine telomeres and telomerase in cellular immortalisation and ageing

To determine the role of telomeres in cellular ageing in equids, we analysed telomere lengths in peripheral blood derived DNA samples from a panel of donkeys (Equus asinus) ranging from 2 to 30 years of age. The average telomere lengths ranged from 7 to 21 kbp and a statistically significant inverse correlation between telomere lengths and donor age was demonstrated. Similarly, telomere lengths in primary fibroblasts isolated from a horse (Equus equus) demonstrated telomeric loss with in vitro ageing when cultured to senescence. We extended this study to evaluate activity of the enzyme telomerase in various equine cell cultures, normal equine tissues and equine benign tumour samples. Initially a panel of equine immortalised and primary cell cultures were evaluated for telomerase activity using a standard telomere repeat amplification protocol (TRAP) assay. High levels of telomerase activity were detected in equine immortalised cells with no activity evident in primary cell cultures. Similarly, no telomerase activity could be detected in normal equine tissues or equine benign tumour samples of the sarcoid or papilloma type. We conclude that telomere attrition may contribute to ageing in equids. However, it would appear that telomerase does not play a major role in the development of the most common benign tumours of the horse.     

大肠癌中端粒长度与DCC基因mRNA表达的研究

目的 分析端粒长度及DCC基因mRNA表达在大肠癌及腺瘤发生发展中的作用。方法 采用Sourthern印迹杂交及RT-PCR技术,分别检测46例大肠腺瘤及62例大肠癌组织中的端粒限制性片段（TRF）长度及DCCmRNA表达状态并观察它们与肿瘤临床病理的关系。结果 在大肠癌及大肠腺瘤中,TRF长度较正常组织明显缩短,其缩短者占53.2%和41.3%,而延长者仅占6.5%和4.4%,结肠癌的TRF长度也较直肠癌的TRF长度明显缩短,DCCmRNA表达缺失率在大肠癌及大肠腺瘤中则分别达62.9%和34.8%,显著高于正常组织（1.6%);同时,大肠癌患者的平均TRF长度还随患者年龄的增长而缩短,DCCmRNA表达缺失率则随肿瘤的分化程度下降及临床阶段的进展而升高,但DCCmRNA表达缺失与TRF长度缩短在大肠癌中未表现出明显的相关性。结论 端粒缩短与DCCmRNA表达缺失与TRF长度缩短在大肠癌中未表现出明显的相关性。结论 端粒缩短与DCCmRNA表达缺失可能是大肠腺瘤恶变及大肠癌形成过程中较具特征表现的生物学异常行为。     

端粒酶在子宫内膜癌组织及其外周血中的表达

目的：探讨端粒酶（telomerase)在子宫内膜组织、外周血中表达与子宫内膜癌发病的关系及其相关因素。方法：采用聚合酶链反应－银染色法（PCR-A)检测端粒酶在32例子宫内膜癌患者和16例正常对照子宫内膜组织及外周血标本中表达情况。结果：32例子宫内膜癌标本,总阳性率94％。外周血表达阳性率62％。癌组织中端粒酶表达明显高于外周血（P＜0．05）。16例正常子宫内膜组织标本中,表达总阳性率为50％（8／16）。其外周血表达总阳性率为25％（6／16）。正常对照两种标本端粒酶表达未显著水平（P＞0．05）。癌患者两种标本总阳性率均明显高于对照组（P＜0．05）。结论：端粒酶表达与子宫内膜癌的发生有密切的关系,同时也受子宫内膜周期性变化的影响,有可能成为肿瘤的标记物。     

Telomere-mediated genomic instability and the clinico-pathological parameters in breast cancer

A study was undertaken to correlate telomere dysfunction and genomic instability with the histopathological grades and the estrogen and progesterone receptor status in breast cancer. Sixty-one archived breast tissues (38 cancer tissues and 23 paired normal tissues) were used in the study. The breast tumor tissues showed significantly shorter telomeres (7.7 kb) compared with the paired adjacent tissues (9.0 kb) by Southern blot analysis. Moreover, telomere shortening was more significant in Grade III tumors than in the Grade II tumors (P = 0.05). Quantitative fluorescence in situ hybridization on paraffin tissue sections revealed a similar trend in telomere shortening. Telomere attrition was associated with telomere dysfunction as revealed by the presence of significantly higher anaphase bridges in tumor cells which was tumor grade dependent. Furthermore, estrogen receptive negative tumors displayed higher anaphase and internuclear bridges. Selected samples from each grade showed greater genomic imbalances in the higher grades than the lower grade tumors as detected by array-comparative genomic hybridization. Telomerase activity was found to be higher in the higher grades (Grade II and III) compared with the lower grade (Grade I). The average mRNA expression of TRF1 and POT1 was lower in the tumor tissues than in the normal tissues. Tankyrase 1 mRNA expression showed a grade-dependent increase in tumor tissues and its expression was also high in estrogen and progesterone negative tumors. The data support the notion that telomere dysfunction might be of value as a marker of aggressiveness of the tumors in breast cancer patients.     

Survivin蛋白表达与胃癌发生发展的关系及意义--组织芯片技术研究

目的探讨凋亡抑制基因Survivin编码蛋白表达与胃癌发生发展的关系及其相关分子病理学机制。方法采用组织芯片仪(M icroarrayer,Beecher Instrum ents,USA)构建含98例胃癌及其癌前病变的直径1.0 mm的225个微组织阵列芯片,免疫组织化学方法检测分析survivin蛋白表达情况。结果胃癌及其癌前病变组织芯片蜡块组织阵列排列整齐,常规切片组织形态良好。凋亡抑制基因survivin编码蛋白在胃癌组织中的表达阳性率显著高于正常胃黏膜、肠上皮化生、不典型增生组织中的表达(P<0.01),胃癌转移组survivin蛋白表达阳性率显著高于无转移组(P<0.05)。Survivin蛋白表达与胃癌病理分期、组织学类型和大体类型没有相关性(P>0.05)。结论组织芯片技术在人胃癌早期诊断中具有其他组织病理学技术不可替代的高效、省时、低消耗的优点。Survivin在胃黏膜癌变过程中被激活而参与胃癌的发生和发展,可作为一个较为理想的肿瘤标志物用于胃癌的早期诊断和转移的预警。     

Telomerase RNA expression during progression of gastric cancer

Telomerase, an enzyme associated with cellular immortality and malignancy, is stringently repressed in most normal somatic cells but is reactivated in malignant tumor cells and immortal cell lines, indicating that activation of telomerase may play an important role in tumorigenesis and immortalization. The pattern of human telomerase RNA (hTR) expression during progression of gastric cancer was investigated by a radioactive in situ hybridization (ISH) assay. Paraffin-embedded sections of 85 archival samples from Korean patients with benign and various malignant stages of gastric carcinomas as well as normal and regenerative tissues were studied. In normal gastric mucosae and regenerative lesions such as chronic peptic ulcer and hyperplastic polyps, only a weak degree of hTR expression was noted, and the expression was limited to basal cells of the gastric glands. Also, a moderate degree of hTR expression was present in the germinal centers of lymphoid follicles present in the submucosa. In tubular adenomas, the degree of hTR expression was also generally weak, but, unlike normal gastric mucosa, the expression was rather diffuse and occasionally focal in distribution. However, moderate to intense and usually diffuse hTR expression was present in all cancerous tissues at different stages. Although some heterogeneity of hTR expression was noted, there was a tendency for intensity of hTR expression to increase gradually as the cancer progressed to a more advanced stage. Our results indicate that upregulation of telomerase expression is associated with gastric cancer development or plays some role in gastric carcinogenesis. Upregulation of hTR expression detected by ISH assay may be a useful marker or tool for the early detection of gastric cancer.     

胃液microRNA-133a在胃癌诊断和预后评估中的作用

目的 探讨胃液中microRNA-133a（MiR-133a）低表达是否与胃癌的进展和预后相关。 方法 采集236例患者的胃液和胃黏膜样品,包括34例健康对照和202例胃癌样本,调查胃液中MiR-133a水平与胃癌的进展以及预后评估之间的相关性。通过Transwell小室实验和划痕实验观察胃癌细胞侵袭和迁移能力的变化。 结果 胃癌患者胃液和癌组织中的MiR-133a水平显著下调,下调幅度与肿瘤分级、T分期和远处转移存在相关性。MiR-133a可显著抑制SGC-7901 胃癌细胞的侵袭和迁移能力。 结论 胃液中MiR-133a低表达与胃癌发展进程和不良临床后果呈现较高相关性,提示胃液中MiR-133a水平可以作为该疾病的进展和预后的鉴别指标。