卵巢癌及癌旁组织端粒酶活性与凋亡相关性研究

目的研究端粒酶活性和抗凋亡基因Bcl-2及促凋亡基因Bax在卵巢癌发生、发展过程中的变化。方法应用SP免疫组织化学方法,对50例卵巢癌及癌旁正常组织进行hTERT、Bcl-2、Bax基因蛋白的检测。结果hTERT和Bcl-2阳性表达率在卵巢癌组织中高于癌旁组织。而Bax阳性表达率在卵巢癌组织低于癌旁组织,2组间均有明显差异。hTERT蛋白表达阳性率在临床Ⅰ、Ⅱ期和临床Ⅲ、Ⅳ期分别为16/23(69.56%)和26/27(96.30%),差异有显著性(P<0.05)。高分化组织与低分化组织中阳性率分别为18/23(78.26%)和25/27(92.59%),差异也有显著性(P<0.05)。对于Bcl-2蛋白表达阳性率在临床Ⅰ、Ⅱ期和Ⅲ、Ⅳ期中分别为8/23(34.78%)和16/27(59.26%),高分化组织和低分化组织阳性率分别为9/23(39.13%)和18/27(66.67%),差异均有显著性(P<0.05)。卵巢癌组织凋亡指数明显低于卵巢癌旁组织,差异有显著性(P<0.05)。结论凋亡调控功能失调与肿瘤的形成有关,hTERT、Bcl-2、Bax基因蛋白的异常表达与卵巢癌发生有关;hTERT、Bcl-2在卵巢癌的发生发展中可能有协同作用,hTERT、Bcl-2与Bax在卵巢癌的发生发展中可能有拮抗作用。hTERT、Bcl-2蛋白的表达可能是反映卵巢癌生物学行为的重要参数。     

卵巢癌及癌旁组织端粒酶活性与凋亡相关性研究

目的研究端粒酶活性和抗凋亡基因Bel-2及促凋亡基因Bax在卵巢癌发生、发展过程中的变化。方法应用SP免疫组织化学方法,对50例卵巢癌及癌旁正常组织进行hTERT、Bcl-2、Bax基因蛋白的检测。结果hTERT和Bcl-2阳性表达率在卵巢癌组织中高于癌旁组织。而Bax阳性表达率在卵巢癌组织低于癌旁组织,2组间均有明显差异。hTERT蛋白表达阳性率在临床Ⅰ、Ⅱ期和临床Ⅲ、Ⅳ期分别为16／23（69．5696）和26／27（96．30％）,差异有显著性（P〈0．05）。高分化组织与低分化组织中阳性率分别为18／23（78．26％）和25／27（92．59％）,差异也有显著性（P〈0．05）。对于Bcl-2蛋白表达阳性率在临床Ⅰ、Ⅱ期和Ⅲ、Ⅳ期中分别为8／23（34．78％）和16／27（59．26％）,高分化组织和低分化组织阳性率分别为9/23（39．13％）和18／27（66．67％）。差异均有显著性（P〈0．05）。卵巢癌组织凋亡指数明显低于卵巢癌旁组织,差异有显著性（P〈0．05）。结论凋亡调控功能失调与肿瘤的形成有关,hTERT、Bel-2、Bax基因蛋白的异常表达与卵巢癌发生有关;hTERT、Bel-2在卵巢癌的发生发展中可能有协同作用,hTERT、Bel-2与Bax在卵巢癌的发生发展中可能有拮抗作用。hTERT、Bel-2蛋白的表达可能是反映卵巢癌生物学行为的重要参数。     

Telomere/telomerase interplay in virus‐driven and virus‐independent lymphomagenesis: pathogenic and clinical implications

Telomerase is a ribonucleoprotein complex critically involved in extending and maintaining telomeres. Unlike the majority of somatic cells, in which hTERT and telomerase activity are generally silent, normal lymphocytes show transient physiological hTERT expression and telomerase activity according to their differentiation/activation status. During lymphomagenesis, induction of persistent telomerase expression and activity may occur before or after telomere shortening, as a consequence of the different mechanisms through which transforming factors/agents may activate telomerase. Available data indicate that the timing of telomerase activation may allow the distinction of two different lymphomagenetic models: (i) an early activation of telomerase via exogenous regulators of hTERT, along with an increased lymphocyte growth and a subsequent selection of cells with increased transforming potential may characterize several virus‐related lymphoid malignancies; (ii) a progressive shortening of telomeres, leading to genetic instability which favors a subsequent activation of telomerase via endogenous regulators may occur in most virus‐unrelated lymphoid tumors. These models may have clinically relevant implications, particularly for the tailoring of therapeutic strategies targeting telomerase. © 2010 Wiley Periodicals, Inc. Med Res Rev     

纤维支气管镜活组织端粒酶活性检测对肺癌的诊断价值

目的探讨肺癌组织端粒酶活性检测对肺癌的诊断价值。方法采用PCR-ELISA法检测了87例纤维支气管镜(纤支镜)活组织的端粒酶活性,其中经病理证实为肺癌病变组织38例,炎症病变组织26例,正常黏膜组织23例。结果肺癌病变组织端粒酶活性水平均值明显高于炎症组织和正常组织(P<0.001),但炎症组织端粒酶活性水平与正常组织比较差异无显著性(P>0.05);不同病理类型肺癌的端粒酶活性水平比较无明显差异(P>0.05)。肺癌病变组织不同病理分级间端粒酶活性比较有显著差异(P<0.05)。结论端粒酶活性检测,对肺癌诊断有重要意义。     

Telomerase as a novel and potentially selective target for cancer chemotherapy

Telomeres are the structures that protect eukaryotic chromosomes from recognition by DNA damage surveillance mechanisms and are maintained in the germ line of multicellular animals by telomerase. In most human somatic cells telomerase is silenced during development and after extensive cell division telomeres shorten to trigger growth arrest. Around 80% of human cancers escape from this growth arrest by re‐activating telomerase but at diagnosis many cancers still have very short telomeres making them very vulnerable to the inhibition of telomerase. As normal cells have a considerable telomere reserve, even in elderly humans, this makes telomerase an attractive and potentially selective anti‐cancer drug target. Proof‐of‐principle experiments are reviewed which show that this optimism may be justified at least for the subset of human cancers with short telomeres. I also address many of the commonly raised concerns that surround telomerase as a target for anti‐cancer drug design.     

Telomerase activity in needle biopsies from prostate cancer and benign prostates

BACKGROUND: Telomerase activation is thought to be essential for the immortality of cancer cells. It may be a prognostic factor in small volume well differentiated prostate cancers and hence a guide for the aggressiveness of the approach. The length of the chromosome tips (telomeres) are maintained by a specific enzyme (telomerase) independently of the normal cell division cycle. Although telomerase is not expressed in most normal human tissues, it is expressed in most human tumours. For the detection of telomerase in small prostate needle biopsy samples a recently developed telomeric repeat amplification protocol (TRAP) assay was used. The aim of the present study was: to measure telomerase activity in human prostate samples, and to evaluate the applicability of this assay on specimens from a prostate biopsy. MATERIALS AND METHODS: From 36 patients referred for lower urinary tract symptoms (LUTS) or suspicion of having prostate cancer a total of 288 prostate biopsy samples were obtained (8 in each patient). When the digital rectal examination was abnormal and/or when the PSA level was elevated in L.U.T.S., or asymptomatic patients' tissue samples were obtained by transrectal ultrasound (TRUS) guided biopsies. Samples were tested for telomerase activity by a modified TRAP and forwarded for histology. RESULTS: In 19 out of 36 patients prostate cancer was diagnosed on histology. In 11 of these 19 tumours substantial telomerase activity was detected, whereas only very low telomerase activity existed in 2 of 17 samples from benign prostatic hypertrophy (BPH) patients. In this small series the relative telomerase activity in prostate cancer correlated with histopathological grade. CONCLUSIONS: Our results show the applicability of a TRAP assay to measure telomerase activity in small needle biopsied prostate samples. In poorly differentiated and metastatic cancer we observed that levels of telomerase activity were high. To establish accuracy and to distinguish the 'relative good from the ugly' further study is needed.     

Targeting lactate metabolism for cancer therapeutics

Lactate, once considered a waste product of glycolysis, has emerged as a critical regulator of cancer development, maintenance, and metastasis. Indeed, tumor lactate levels correlate with increased metastasis, tumor recurrence, and poor outcome. Lactate mediates cancer cell intrinsic effects on metabolism and has additional non–tumor cell autonomous effects that drive tumorigenesis. Tumor cells can metabolize lactate as an energy source and shuttle lactate to neighboring cancer cells, adjacent stroma, and vascular endothelial cells, which induces metabolic reprogramming. Lactate also plays roles in promoting tumor inflammation and in functioning as a signaling molecule that stimulates tumor angiogenesis. Here we review the mechanisms of lactate production and transport and highlight emerging evidence indicating that targeting lactate metabolism is a promising approach for cancer therapeutics.     

质粒介导RNAi靶向hTERT抑制K562细胞端粒酶活性的研究

本研究探讨质粒载体介导的RNAi靶向hTERT对白血病细胞株K562 hTERT基因封闭、端粒酶活性抑制的作用。针对hTERT mRNA化学合成3条siRNA链转染K562细胞,筛选2条高效特异的siRNA链,构建靶向hTERT mRNA的质粒载体pSUPER-U6-Kanr-hTERT-1、pSUPER-U6-Kanr-hTERT-2,在脂质体介导下转染K562细胞;48、72小时后分析靶基因hTERT mRNA的表达量,检测细胞端粒酶活性,同时检测细胞凋亡率。结果表明：3条siRNA链中,48小时目的基因表达抑制,但抑制率有差异,72小时后抑制作用渐消失;转染质粒pSUPER-U6-Kanr-hTERT-1（P-1组）、pSUPER-U6-Kanr-hTERT-2（P-2组）的K562细胞hTERT mRNA表达均下降,P-1组48小时时为0.39±0.13,72小时时为0.57±0.32,P-2组48小时时为0.55±0.20,72小时时为0.88±0.23;端粒酶活性P-1组48小时时为0.42±0.07,72小时时为0.31±0.08;P-2组48小时时为0.49±0.27,72小时时为0.39±0.03;两组均较阴性对照明显下降,且以P-1组作用明显。48小时细胞凋亡率P-1组为18.39±3.08%,P-2组为15.5±3.59%,与阴性对照组7.64±3.73%相比有显著性差异,72小时细胞凋亡率P-1组为13.2±1.18%、P-2组为12.86±3.09%,与阴性对照组8.07±0.19%相比无统计学差异。结论：靶向hTERT的RNAi可抑制目的基因hTERT mRNA表达,进而抑制端粒酶活性。此抑制作用与靶位点选择密切相关,实验中si-hTERT-1优于si-hTERT-2;si-hTERT-3几乎无作用。由质粒载体介导RNAi作用时间明显长于化学合成siRNA,前者72小时甚至更长,后者仅48小时。端粒酶活性被抑制后,48小时的细胞凋亡较对照组有所增加,72小时时与对照组无差别,推测端粒酶活性下调后部分细胞可能被诱导分化。     

食管癌组织和切缘端粒酶活性表达的研究

目的:研究食管癌组织和切缘端粒酶活性表达的意义及其与预后的关系。方法:应用RT-PCR技术检测80例食管癌和10例食管良性肿瘤组织中及切缘(近心端切缘病理切片无癌组织残留)的端粒酶活性。结果:在80例食管癌组织中端粒酶活性表达为82.5%(66例),切缘端粒酶活性表达为12.5%(10例)。10例食管良性肿瘤组织中及切缘均未发现端粒酶阳性表达。癌组织端粒酶活性与食管癌TNM分期、病理分化程度、淋巴结转移及预后有显著性相关。切缘端粒酶活性与食管癌TNM分期、肿瘤部位有显著性相关。术后随访6个月～3年,共有9例胃吻合口复发,其中8例为术后食管切缘端粒酶活性阳性患者。结论:端粒酶活性表达与食管癌进展与预后有关,检测食管切缘端粒酶活性可作为早期预测食管癌术后吻合口复发的重要依据。     

宫颈上皮内瘤变中hTERC基因扩增状况的研究

目的 探讨人染色体端粒酶基因(hTERC)在宫颈病变中的表达及其临床意义.方法 采用荧光原位杂交方法 检测20例正常宫颈和100例有不同程度宫颈病变的脱落细胞学标本及10例宫颈鳞癌组织标本中hTERC基因的表达情况.结果 hTERC基因在正常组、ASCUS、ASCUS-H、LSIL和HSIL组中的阳性表达率分别为0、19.44%、57.14%、41.86%和71.43%;在正常组、CIN1、CIN2、CIN3和宫颈鳞癌组中的阳性表达率分别为12.5%、60%、66.67%、75%和100%.hTERC基因在正常组与上皮内瘤变各组以及与宫颈鳞癌组组间比较差异显著.结论 hTERC基因高表达在宫颈癌发生、发展中可能起重要作用,采用FISH检测hTERC基因有望成为宫颈癌早期筛查的方法 之一.     

Erosion of telomeric single-stranded overhang in patients with aplastic anaemia carrying telomerase complex mutations

Background  Loss-of-function mutations in telomerase complex genes reduce telomerase activity and shorten overall telomere length in leucocytes, and they can clinically manifest as bone marrow failure (aplastic anaemia and dyskeratosis congenita) and familial pulmonary fibrosis. Telomeres are constituted of double-stranded tandem TTAGGG repeats followed by a 3' G-rich single-stranded overhang, a crucial telomeric structural component responsible for the t-loop formation.
Materials and methods  We investigated the length of telomeric overhangs in 25 healthy individuals from 0 to 76 years of age, 16 patients with aplastic anaemia, and 13 immediate relatives using a non-denaturing in-gel method and the telomere-oligonucleotide ligation assay.
Results  Telomeric overhang lengths were constant from birth to eighth decade of life in healthy subjects, in contrast to overall telomere length, which shortened with ageing. Most patients with marrow failure and a telomerase gene mutation showed marked erosion of telomeric overhang associated with critically short telomeres; in other aplastic patients with normal genotypes, normal overall telomere lengths and who responded to immunosuppressive therapy, telomeric overhangs were maintained.
Conclusions  Telomeric overhang erosion does not participate in physiological ageing but support a role for eroded telomeric overhangs and abnormal telomere structure in pathological shortening of telomeres, especially caused by loss-of-function telomerase mutations. Disrupted telomere structure caused by short telomeric overhangs may contribute to the mechanisms of abnormal haematopoietic compartment senescence and chromosomal instability in human bone marrow failure.     

端粒酶基因在胃癌前病变的表达

目的　探讨端粒酶基因与胃癌前病变恶性转化的关系及其作用。方法　应用原位杂交方法检测端粒酶基因hTRT在113例不同病变胃粘膜组织的表达。结果　在慢性浅表性胃炎(CSG)、慢性萎缩性胃炎(CAG)、胃息肉(GP)、残胃(GR)、胃溃疡(GU)和胃癌(GC)中端粒酶hTRT的阳性表达率分别为0 % (0 / 2 3)、2 6 . 6 8% (4/ 15 )、0 % (0 / 15 )、7 6. 9% (1/ 13)、14 2 9(3/ 2 1)和80 . 77% (2 1/ 2 6 )。结论　端粒酶基因hTRT的表达与胃癌前病变的恶性转化密切相关,其重新激活可能在胃癌的发生中起关键作用。     

端粒酶PCR SCE检测对诊断膀胱癌的价值及应用

目的 探明膀胱癌患者端粒酶阳性率及姊妹染色单体互换（SCE）率，以确定其诊断及应用价值。方法 采用膀胱癌及对照正常膀胱的组织进行端粒酶活性检测（TRAP-银染法）、外周血淋巴细胞SCE检测，然后进行比较分析。结果 （1）组织标本的端粒酶阳性率：膀胱癌93．75％（30／32），正常膀胱组织0％（0／30），表明膀胱癌端粒酶高度表达，其与正常对照组比较差异有显著性（P〈0．001）。（2）外周血淋巴细胞SCE率（X）：膀胱癌32例（10．06），膀胱癌的SCE率均显著高于正常对照组（P〈0．001）；（3）膀胱癌患者的端粒酶活性与SCE率的关系：阳性组32例SCE率10．06；膀胱癌患者端粒酶阴性组30例SCE率9．17（P〈0．05）。结论 端粒酶PER及SCE检测对于膀胱癌诊断及鉴别良恶性均具有重要的价值，可作为辅助诊断及攀别诊断指标．SCE率可作为膀胱肿瘤的疗效评价指标。     

人胃癌细胞系端粒酶逆转录酶基因启动子区域甲基化的检测

目的研究人胃癌细胞系端粒酶逆转录酶基因（hTERT）启动子区域是否存在甲基化,甲基化程度是否与肿瘤的恶性程度相关。方法运用甲基化特异性PCR（MSP）方法,以人乳腺癌细胞、人胚肺成纤维细胞作为阳性及阴性对照,检测中、低分化的胃腺癌细胞系hTERT基因启动子区域甲基化情况。结果端粒酶阳性的胃癌细胞、人乳腺癌细胞均存在甲基化,而端粒酶阴性的人胚肺成纤维细胞不存在甲基化,不同分化程度胃癌细胞的hTERT启动子区CpG位点甲基化情况不同。结论hTERT基因启动子区域的甲基化,与细胞的分化程度相关,可能参与了胃癌细胞的发生与发展。