Testicular expression of survivin and human telomerase reverse transcriptase(hTERT)associated with spermatogenic function in infertile patients

Aim： To characterize the coexpression of survivin, an inhibitor of apoptosis （IAF）, and human telomerase reverse transcriptase （hTERT） in human testes with varying spermatogenic function. Methods： Transcript levels of survivin mRNA and hTERT mRNA were determined in normal testes （n = 11） and testes with defective spermatogenesis （n = 28） using real-time reverse-transcription polymerase chain reaction （RT-PCR）. The histological work-up was performed according to a modified Johnsen score. Results： Expressions of both survivin and hTERT were highest at median levels of 96.8 and 709 in normal spermatogenesis and dropped to 53.3 and 534 in testes with postmeiotic spermatogenic arrest （n = 10）. In severe spermatogenic failure （n = 18）, survivin expression was lacking in most specimens （n = 16）, whereas at least low levels of testicular hTERT expression were largely detectable with a normalized expression of 73 in premeiotic spermatogenic arrest （n = 7） and 45 in patients with Sertoli cell-only syndrome （SCOS） （n = 3）. Both survivin and hTERT expressions increased with a progressing Johnsen score （P for trend = 0.001）. Conclusion： Although both survivin and hTERT are correlated with spermatogenic function, they show different expression patterns in testes of infertile patients. These findings substantiate results from studies in the rodent testis suggesting a predominant expression of survivin in meiotically dividing germ cells. （Asian J Andro12006 Jan; 8： 95-100）

Quantification of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) mRNA in testicular tissue of infertile patients

Aim: To evaluate the quantitative detection of human telomerase RNA (hTR) and human telomerase reverse tran-scriptase (hTERT) mRNA as diagnostic parameters in the workup of testicular tissue specimens from patients presentingwith non-obstructive azoospermia. Methods: hTR and hTERT mRNA expression were quantified in 38 cryopre-served testicular tissue specimens by fluorescence real-time reverse transcription - polymerase chain reaction (RT-PCR)in a LightCycler(r). This was paralleled by conventional histological workup in all tissue specimens and additionalsemithin sectioning preparation in cases with maturation arrest (n = 12) and Sertoli-cell-only syndrome (n = 12). Re-sults; The average normalized hTERT expression (N_(hTERT)) was 131.9±48.0 copies (mean ± SD) in tissue speci-mens with full spermatogenesis, N_(hTERT) = 51.2 ±17.2 copies in those with maturation arrest and N_(hTERT) = 2.7 ± 2.4copies in those with Sertoli-cell-only syndrome (SCOS). The discriminant analysis showed that detection of N_(hTERT)(N_(hTR)) had a predictive value of 86.8% (55.3%) for correct classification in one of the three histological subgroups.Conclusion; Our results demonstrate that quantitative detection of hTERT mRNA expression in testicular tissue en-ables a molecular-diagnostic classification of gametogenesis. Quantitative detection of hTERT in testicular biopsies isthus well suited for supplementing the histopathological evaluation. (Asian J Androl 2001 Dec; 3: 263 - 270)

Neuroprotective effects of human telomerase reverse transcriptase on beta-amyloid fragment 25-35-treated human embryonic cortical neurons

BACKGROUND： Numerous current studies have suggested that human telomerase reverse transcriptase （hTERT） gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the mechanism of action remains unknown. OBJECTIVE： To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 （AI325-35）. DESIGN, TIME AND SETTING： The randomized, controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research, Zhongshan School of Medicine, Sun Yat-sen University, China, from September 2005 to June 2008. MATERIALS： AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University, China. Human cortical neurons were derived from 12-20 week old aborted fetuses, obtained from the Guangzhou Maternal and Child Health Hospital, China. Mouse anti-Odk5 and mouse anti-p16 monoclonal antibodies （Lab Vision, USA）, and mouse anti-hTERT monoclonal antibody （Epitomics, USA）, were used in this study. METHODS： （1） Recombinant adenovirus vectors, encoding hTERT （Ad-hTERT） and green fluorescent protein （Ad-GFP）, were constructed using the AdEasy-1 Expression System. Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise, human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days. Human embryonic cortical neurons in the control group were cultured as normal. （2） Human embryonic cortical neurons in the Ad-hTERT group were treated with 10 pmol/L Aβ25-35 for 24 hours. Normal human embryonic cortical neurons treated with 10 pmol/Lβ25.35 for 24 hours served as a model group. Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ25-35. MAIN OUTCOME MEASURES： Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol （TRAP） ELISA kit. Neural activity in human embryonic cortical neurons was examined by MTT assay; apoptosis was measured using TUNEL assay; and Cdk5 and p16 protein expressions were measured by Western blot. RESULTS： Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group. No hTERT expression was detected in the Ad-GFP and control groups. Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups （P 〈 0.01）. Compared with the control group, cell activity was significantly decreased （P 〈 0.05）, and cell apoptotic rate, Cdk5 and p16 expression were significantly increased （P 〈 0.01） in the model group. Compared with the model group, cell activity was increased in the Ad-hTERT group, and peaked at day 3 post-transfection （P 〈 0.05）. Neuroprotective effects also peaked at day 3 post-transfection; and the apoptotic rate, Cdk5 and p16 expression significantly decreased （P 〈 0.01）. CONCLUSION： Expression of hTERT in human embryonic cortical neurons can relieve Aβ25-35-induced neuronal apoptosis. The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.

Inhibition of telomerase with human telomerase reverse transcriptase antisense increases the sensitivity of tumor necrosis factor-α-induced apoptosis in prostate cancer cells

Aim： To investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase （hTERT） antisense on tumor necrosis factor-α （TNF-α-induced apoptosis in prostate cancer cells （PC3）. Methods： Antisense phosphorothioate oligodeoxynucleotide （AS PS-ODN） was synthesized and purified. Telomerase activity was measured using the telomeric repeat amplification protocol （TRAP） and polymerase chain reaction enzyme-linked immunoassay （PCR-ELISA）. hTERT mRNA was measured by reverse transcription PCR （RT-PCR） assay and gel-image system, hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was detected by 3-（4, 5-dimethylthiazol-2-yl）-2,5-Diphenyltetrazolium （MTT） assay. Cell apoptosis was observed by morphological method and determined by flow cytometry. Results： The telomerase activity decreased with time after hTERT AS PS-ODN treatment. The levels of hTERT mRNA decreased with time after hTERT AS PS-ODN treatment, which appeared before the decline of the telomerase activity. The percentage of positive cells of hTERT protein declined with time after hTERT AS PS-ODN treatment, which appeared after the decline of hTERT mRNA. There was no difference in telomerase activity, hTERT mRNA and protein levels between hTERT sense phosphorothioate oligodeoxynucleotide （S PS-ODN） and the control group. The cell viability decreased with time after hTERT AS PS-ODN combined with TNF-α treatment. The percentage of apoptosis increased with time after hTERT AS PS-ODN combined with TNF-α treatment. There was no difference in cell viability and the percentage of apoptosis between hTERT S PS-ODN and the control group. Conclusion： hTERT AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression, and inhibition of telomerase with hTERT antisense can enhance TNF-α- induced apoptosis of PC3 cells.

Unique case of oligoastrocytoma with recurrence and grade progression:Exhibiting differential expression of high mobility group-A1 and human telomerase reverse transcriptase

Mixed gliomas, primarily oligoastrocytomas, account for about 5%-10% of all gliomas. Distinguishing oligoastrocytoma based on histological features alone has limitations in predicting the exact biological behavior, necessitating ancillary markers for greater specificity. In this case report, human telomerase reverse transcriptase(hT ERT) and high mobility group-A1(HMGA1); markers of proliferation and stemness, have been quantitatively analyzed in formalin-fixed paraffin-embedded tissue samples of a 34 years old patient with oligoastrocytoma. Customized florescence-based immunohistochemistry protocol with enhanced sensitivity and specificity is used in the study. The patient presented with a history of generalized seizures and his magnetic resonance imaging scans revealed infiltrative ill-defined mass lesion with calcified foci within the left frontal white matter, suggestive of glioma. He was surgically treated at our center for four consecutive clinical events. Histopathologically, the tumor was identified as oligoastrocytoma-grade Ⅱ followed by two recurrence events and final progression to grade Ⅲ. Overall survival of the patient without adjuvant therapy was more than 9 years. Glial fibrillary acidic protein, p53, Ki-67, nuclear atypia index, pre-operative neutrophillymphocyte ratio, are the other parameters assessed. Findings suggest that hT ERT and HMGA1 are linked to tumor recurrence and progression. Established markers can assist in defining precise histopathological grade in conjuction with conventional markers in clinical setup.

Expression of Telomerase Reverse Transcriptase during the Malignant Transformation of Cadmium-Induced Cells

The objective of the present study was to investigate human telomerase reverse transcriptase (hTERT) mRNA and protein expressions during the cadmium chloride-induced malignant transformation of human bronchial epithelial (16HBE) cells. Fluorescence quantitative PCR (FQ-PCR) and Western blot analyses were performed to detect the hTERT mRNA and protein expressions in normal 16HBE cells, cadmium chloride-transformed 16HBE cells, and tumorigenic cells from nude mice inoculated with cadmium chloride-transformed 16HBE cells. Under the inner standard of GAPDH, the hTERT mRNA expression was significantly higher at different stages of malignant transformation (cadmium chloride-transformed 16HBE cells at passages 15 and 35 and tumorigenic cells from nude mice) than in normal 16HBE cells, and increased with the development of malignancy (P < 0.01). In addition, hTERT protein expression increased with the development of malignancy. These findings demonstrate that hTERT expression is related to cadmium chlorideinduced malignant transformation. Cadmium chloride-induced malignant transformation is involved in changes in the hTERT activity, and might be an early event in cadmium chloride-induced malignant transformation.

子痫前期孕妇血清hTERT和Sirt6水平表达与疾病严重程度及妊娠结局评估中的价值研究

目的检测子痫前期孕妇血清中人端粒酶反转录酶(human telomerase reverse transcriptase,hTERT)、沉默信息调节因子6(silent information regulator 6,Sirt6)表达,并探究hTERT,Sirt6水平表达与疾病严重程度及妊娠结局评估中的价值。方法选取2018年1月~2022年12月在陕西省人民医院进行诊治的300例子痫前期孕妇作为子痫前期组,孕妇均符合《妊娠期高血压疾病诊治指南(2015)》中子痫前期诊断标准,选取同时期孕检的300例健康孕妇为对照组,根据病情严重程度将子痫前期组分为轻症子痫前期组(n=180)和重症子痫前期组(n=120),根据是否发生不良妊娠结局将子痫前期组分为正常妊娠组(n=165)和不良妊娠组(n=135)。酶联免疫吸附实验(enzyme-linked immunosorbnent assay,ELISA)法检测血清中hTERT和Sirt6水平,Spearman相关性分析血清中hTERT和Sirt6水平与子痫前期孕妇病情严重程度的相关性,利用受试者工作特征(receiver operating characteristic,ROC)曲线评估血清hTERT和Sirt6水平在子痫前期诊断及妊娠结局预测中的价值。结果与对照组比较,子痫前期组血清hTERT(22.15±5.82 ng/ml vs 30.12±9.56 ng/ml),Sirt6(5.26±1.62 ng/ml vs 7.06±2.29 ng/ml)水平降低,差异具有统计学意义(t=12.334,11.114,均P<0.001)。与轻症子痫前期组比较,重症子痫前期组孕妇血清hTERT(18.28±4.11 ng/ml vs 24.73±6.96 ng/ml),Sirt6(4.03±1.17 ng/ml vs 6.08±1.92 ng/ml)水平降低,差异具有统计学意义(t=9.142,10.469,均P<0.001)。与正常妊娠组比较,不良妊娠组子痫前期孕妇血清中hTERT(17.75±4.61 ng/ml vs 25.75±6.81 ng/ml),Sirt6(4.06±0.96 ng/ml vs 6.24±2.16 ng/ml)水平降低,差异具有统计学意义(t=11.639,10.878,均P<0.001)。Spearman相关性分析显示,血清hTERT,Sirt6水平与子痫前期孕妇疾病严重程度均呈负相关(r=-0.562,-0.604,均P<0.001)。ROC曲线分析结果显示,血清hTERT,Sirt6诊断子痫前期的曲线下面积(95%置信区间)[AUC(95%CI)]分别为0.711(0.673~0.747),0.727(0.689~0.762),两者联合诊断子痫前期的AUC(95%CI)为0.788(0.753~0.820),高于两者单独诊断(Z=2.719,2.154,P=0.007,0.031);血清hTERT,Sirt6预测子痫前期不良妊娠结局的AUC(95%CI)分别为0.786(0.735~0.831),0.783(0.732~0.829),两者联合预测子痫前期不良妊娠结局的AUC(95%CI)为0.849(0.804~0.888),高于两者单独预测(Z=1.855,1.861,P=0.032,0.031)。结论hTERT和Sirt6在子痫前期孕妇血清中水平较低,与子痫前期孕妇疾病严重程度均呈负相关,并对妊娠结局具有一定的评估价值。

Inhibitory Effects of Selenium on Telomerase Activity and hTERT Expression in Cadmium-transformed 16HBE Cells

To investigate the effects of sodium selenite on telomerase activity and expression of hTERT mRNA in cadmium-transformed 16HBE cells. Methods Telomerase activity and expression of genes were measured after cultured cadmium-transformed 16HBE cells were exposed to sodium selenite at different doses （0.625, 1.25, 2.50, 5.00 pmol/L） for 24 hours. Results Selenium decreased telomerase activity in cadmium-transformed 16HBE cells. There existed an obvious dose-effect relationship between the selenium concentration and these changes. The expression of hTERT and c-myc mRNA also decreased but the expression of madl mRNA increased after exposure to selenium for 24 hours. No difference was found in expression of hTRF1 and hTRF2 mRNA after incubated with sodium selenite for 24 hours, compared with control group. Conclusion Selenium inhibits telomerase activity by decreasing hTERT and c-myc mRNA expression and increasing madl mRNA expression in cadmium-transformed 16HBE cells and selenium concentration is significantly correlated with these changes.

Telomerase and hTERT: Can they serve as markers for gastric cancer diagnosis?

AIM:To investigate telomerase activity and human telomerase reverse transcriptase(hTERT)expression in normal human gastric mucosal epithelial cells(nhGMECs)and fibroblasts(nhGMFs).METHODS:nhGMECs and nhGMFs were isolated and cultured from specimens obtained during routine surgery for bleeding peptic ulcer.Telomerase activity in nhGMFs,nhGMECs,and the tumor cell lines BGC-823,SGC-7901 and MKN-28 cells was analyzed using the telomeric repeat amplification protocol assay.hTERT protein was determined in nhGMECs,nhGMFs,BGC-823,SGC-7901 and MKN-28 cells by indirect immunofluorescence.served in nhGMECs,nhGMFs and BGC-823,SGC-7901,MKN-28 cell lines.Positive hTERT immunostaining was detected in nhGMECs,nhGMFs,BGC-823,SGC-7901and MKN-28 cell lines.CONCLUSION:The use of telomerase or hTERT as diagnostic markers for gastric cancer may require further studies.

Inhibition of Cell Growth and Telomerase Activity in Osteosarcoma Cells by DN-hTERT

In order to study the effects of dominant negative human telomerase reverse transcriptase （DN-hTERT） on cell growth and telomerase activity in osteosarcoma cell line MG63, MG63 cells were transfected with DN-hTERT-IRES2-EGFP9 （DN） or IRES2-EGF （I, blank vector） with lipofectamine 2000. The stably transfected cells were selected with G-418. Cell growth properties were examined under a fluorescence microscope. The hTERT mRNA expression was detected by reverse transcription-polymerase chain reaction （RT-PCR）. Telomerase activities were measured by TRAP-ELISE. The tumorigenicity was studied with tumor xenografts by subcutaneous injection of cancer cells into nude mice. The results showed that cell growth was suppressed in MG63 cells transfected with DN-hTERT. The hTERT mRNA was increased in N-hTERT transfected-MG63 cells （MG63/DN）. The telomerase activity was 2.45±0.11 in MG63/DN cells, while 3.40±0.12 in the cells transfected with blank vector （MG63/I）, （P〈0.05）; DN-hTERT-expressing clones did not form tumors in 2 weeks, but the ratio of tumorigenesis was 30 % in nude mice bearing MG63/I （P〈0.01）. It was concluded that DN-hTERT could specifically inhibit the cell growth and telomerase activity in MG63 cells.

Expression of Telomerase Subunits in Gastric Cancer

To detect the expression of telomerase subunits （human telomerase reverse transcriptase, human telomerase associated protein 1 and human telomerase RNA） in gastric cancer and to examine the role that different telomerase subunits play in the gastric carcinogenesis, reverse transcription-polymerase chain reaction （RT-PCR） was used to detect telomerase suhunits messenger RNA in 24 samples of gastric cancer and corresponding non-cancerous tissue. The results showed that the positive rate of hTERT mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 25 %, respectively. The former was significantly higher than the latter （X^2 = 26.4, P〈0.01）. The positive rate of hTEP1 mRNA from gastric cancer and corresponding non-cancerous tissues was 100 % and 91.7%, respectively and no significant difference was found between them （X^2 =2.1, P〉0.05）. The positive rates of hTR for gastric cancer and corresponding non-cancerous tissues were both 100 % and no significant difference existed between them. It is concluded that in contrast to hTEP1 and hTR, the up-regulation of hTERT mRNA expression may play a more important role in the development of gastric cancer.

基于外周血hTERT、UCA1表达的列线图模型对老年膀胱尿路上皮癌术后无病生存的预测价值

目的:探讨基于围术期数据及外周血人端粒酶反转录酶(hTERT)、尿路上皮癌抗原1(UCA1)表达的列线图模型对老年膀胱尿路上皮癌术后无病生存的预测价值。方法:选取2017年4月至2019年4月首都医科大学附属北京友谊医院收治的268例老年膀胱尿路上皮癌患者,均行根治性膀胱切除术,随机分为建模人群和验证人群,根据术后3年无病生存率分为死亡组和存活组,比较两组一般资料和围术期数据。采用实时荧光定量PCR(RT-qPCR)法检测外周血hTERT、UCA1基因表达。采用Lasso、Cox回归方程分析老年膀胱尿路上皮癌术后无病生存的影响因素,构建列线图模型,绘制受试者工作特征曲线(ROC)、校准曲线及决策曲线分析预测性能。结果:患者术后3年无病生存率为55.77%。外周血hTERT、UCA1表达,输尿管切缘、年龄、肿瘤分期和淋巴结转移均为老年膀胱尿路上皮癌术后无病生存的影响因素(均P<0.05),基于上述因素构建的列线图模型在建模人群和验证人群中C-index分别为0.857、0.949,经校准曲线分析,0.2~1.0、0.1~1.0范围内该模型在建模人群和验证人群中预测净获益值较高。结论:外周血hTERT、UCA1高表达为老年膀胱尿路上皮癌患者术后无病生存的危险因素,所构建模型对患者术后无病生存状况有一定的预测价值。

Effects of Combined siRNA-TR and-TERT on Telomerase Activity and Growth of Bladder Transitional Cell Cancer BIU-87 Cells

The effects of combined RNA interference(RNAi) of human telomerase RNA(hTR) and human telomerase reverse transcriptase(hTERT) genes on telomerase activity in a bladder cancer cell line(BIU-87 cells) were investigated by using gene chip technology in vitro with an attempt to evaluate the role of RNAi in the gene therapy of bladder transitional cell cancer(BTCC).Three TR-specific double-stranded small interfering RNAs(siRNAs) and three TERT-specific double-stranded siRNAs were designed to target different regions of TR and TERT mRNA.The phTR-siRNA,phTERT-siRNA,and the combination of both plasmids phTR+phTERT-siRNA were transfected into BIU-87 cells.The expression of hTR and hTERT mRNA was detected by quantitative fluorescent reverse transcription-polymerase chain reaction,and a telomeric repeat amplification protocol was applied to detect telomerase activity.Growth inhibition of BIU-87 cells was measured by MTT assay.Gene chip analysis was performed to evaluate the effects of the combined RNAi of hTR+hTERT genes on telomerase activity and growth of BIU-87 cells in vitro.The results showed that the expression of hTERT and hTR mRNA was inhibited by pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,and pRNAT-hTR-Ⅲ+hTERT-Ⅲ in BIU-87 cells.The inhibition efficiency of pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,pRNAT-hTERT-Ⅲ+pRNAT-hTR-Ⅲ was 67% for TERT mRNA,41% for TR mRNA,57% for TR mRNA and 70% for TERT mRNA in BIU-87 cells respectively.The growth of BIU-87 cells was inhibited and telomerase activity was considerably decreased,especially in the cells treated with combined RNAi-hTR and-hTERT.Gene chip analysis revealed that 21 genes were down-regulated(ATM,BAX,BCL2,BCL2L1,BIRC5,CD44,CTNNB1,E2F1,JUN,MCAM,MTA1,MYC,NFKB1,NFKBIA,NME4,PNN,PNN,SERPINE1,THBS1,TNFRSF1A,and UCC1).The results indicated that hTR-siRNA and hTERT-siRNA,especially their combination,siRNA hTR+hTERT,specifically and effectively suppressed the expression of both hTR and hTERT mRNA and telomerase activity.Molecular biological mechanism by which combined siRNA-TR and-TERT inhibited telomerase activity and growth of BIU-87 cells in vitro may involve the down-regulation of the 21 genes.

Antitumor activity of an hTERT promoter-regulated tumor-selective oncolytic adenovirus in human hepatocellular carcinoma

AIM: To construct a tumor-selective replication-competent adenovirus (RCAd), SG300, using a modified promoter of human telomerase reverse transcriptase (hTERT). METHODS: The antitumor efficacy of SG300 in hepatocellular carcinoma was assessed in vitro and in vivo. In vitro cell viability by MTT assay was used to assess the tumor-selective oncolysis and safety features of SG300, and in vivo antitumor activity of SG300 was assessed in established hepatocellular carcinoma models in nude mice. RESULTS: SG300 could lyse hepatocellular carcinoma cells at a low multiplicity of infection (MOI), but could not affect growth of normal cells even at a high MOI. Both in Hep3B and SMMC-7721 xenograft models of hepatocellular carcinoma, SG300 had an obvious antitumor effect, resulting in a decrease in tumor volume. Its selective oncolysis to tumor cells and safety to normal cells was also superior to that of ONYX-015. Pathological examination of tumor specimens showed that SG300 replicated selectively in cancer cells and resulted in apoptosis and necrosis of cancer cells. CONCLUSION: hTERT promoter-regulated replicativeadenovirus SG300 has a better cancer-selective replication-competent ability, and can specifically kill a wide range of cancer cells with positive telomerase activity, and thus has better potential for targeting therapy of hepatocellular carcinoma.

Inhibitory effects of antisense phosphorothioate oligodeoxynucleotides on pancreatic cancer cell Bxpc-3 telomerase activity and cell growth in vitro

瞄准：在增长和胰腺的癌症房间线 Bxpc-3 的 telomerase 活动上调查 telomerase hTERT 基因反感觉 oligonucleotide (hTERT-ASO ) 的效果。方法：MTT 试金被用来在不同时间在 Bxpc-3 房间的增长上检测 hTERT-ASO 的不同剂量的效果。学习反肿瘤活动，房间被划分成三个组：控制组(胰腺的癌症房间 Bxpc-3 ) ；反察觉到 oligonucleotide (hTERT-ASO ) 组；并且没有意义 oligonucleotide 组与 phosphorothioate 装饰了。Telomerase 活动用 TRAP-PCR-ELISA 被检测。房间 DNA 分发用流动血细胞计数试金被检验。房间 apoptosis 被传播电子显微镜在每个组观察。结果：有 6 mmol/L hTERT-ASO 的术后疗法，房间增长在剂量依赖者和时间依赖者举止被禁止。telomerase 活动减少了有为 72 h 的 hTERT-ASO 的术后疗法。流动血细胞计数证明 G0/G1 阶段的房间数字从 2.7% ～ 14.7% 增加了， S 阶段的房间数字从 72.7% ～ 51.0% 减少了，并且一座 sub-G1 阶段房间 apoptosis 山峰出现在 G1 舞台前面。结论：Telomerase 反感觉 oligodeoxy 核苷酸能禁止胰腺的癌症房间线 Bxpc-3 和减少的增长 telomerase 活动和增加房间 apoptosis 率在试管内。

EXPRESSION OF A TELOMERASE-ASSOCIATED GENE IN NORMAL, ATROPHIC ,AND TUMOROUS TESTES

Objective To evaluate the expression of telomerase transcriptional elements-interacting factor (TEIF) in human testis under different status and its relation with human telomerase reverse transcriptase (hTERT) expression. Methods Specific antisera against TEIF were generated by immunization of rabbits with purified recombinated partial TEIF. Samples were assigned to three groups according to their pathological types, including 16 normal testes, 8 atrophic testes, and 6 testicular seminomas. They were subjected to immunohistochemical staining of TEIF and hTERT. Results from both TEIF and hTERT were analyzed semi-quantitatively and compared. Results The expressions of TEIF and hTERT were detected in all samples of normal, atrophic testes, and seminomas. No differences of TEIF expressions among these three groups were observed (P > 0.05). On the contrary, the expressions of hTERT were significantly lower in atrophic testes compared with those of normal testes and seminomas (both P < 0.05). Nevertheless, co-expressions of TEIF with hTERT were revealed to be in normal and malignant cases (P < 0.05) but not in atrophic testes, which generally presented TEIF expression. The cellular distributions of both proteins were similar and mainly in spermatocytes and some Sertoli cells, while were all negative in the interstitial cells and other stromal cells. Conclusions The uniform expressions of TEIF in all these specimens suggest that it may be a marker of testis and its related diseases. The strong expression of hTERT in normal testes and testicular seminomas comparing with the low expres- sion in atrophic testes may suggest a role for telomerase in maintaining proliferation of germ cells.

Telomerase activity: An attractive target for cancer therapeutics

Telomeres are non-coding tandem repeats of 1000-2000 TTAGGG nucleotide DNA sequences on the 3' termini of human chromosomes where they serve as protective "caps" from degradation and loss of genes. The "cap" at the end of chromosome required to protect its integrity is a 150-200 nucleotide-long single stranded G-rich 3' overhang that forms two higher order structures, a T-loop with Sheltering complex, or a G-quadruplex complex. Telomerase is a human ribonucleoprotein reverse transcriptase that continually added single stranded TTAGGG DNA sequences onto the single strand 3' of telomere in the 5' to 3' direction. Telomerase activity is detected in male germ line cells, proliferative cells of renewal tissues, some adult pluripotent stem cells, embryonic cells, but in most somatic cells is not detected. Re-expression or up-regulation of telomerase in tumours cells is considered as a critical step in cell tumorigenesis and telomerase is widely considered as a tumour marker and a target for anticancer drugs. Different approaches have been used in anticancer therapeutics targeting telomerase. Telomerase inhibitors can block directly Human TElomerase Reverse Transcriptase(h TERT) or Human TElomerase RNA telomerase subunits activity, or G-quadruplex and Sheltering com-plex components, shortening telomeres and inhibiting cell proliferation. Telomerase can become an immune target and GV1001, Vx-001, I540 are the most widespread vaccines used with encouraging results. Another method is to use h TERT promoter to drive suicide gene expression or to control a lytic virus replication. Recently telomerase activity was used to activate pro-drugs such as Acycloguanosyl 5'-thymidyltriphosphate, a synthetic ACV-derived molecule when it is activated by telomerase it does not require any virus or host active immune response to induce suicide gene therapy. Advantage of all these therapies is that target only neoplastic cells without any effects in normal cells, avoiding toxicity and adverse effects of the current chemotherapy. However, as not all the approaches are equally efficient, further studies will be necessary.

EXPRESSION OF A MUTANT hTERT IN HUMAN BLADDER CARCINOMA CELL LINE T24 AND ITS CLINICAL SIGNIFICANCE

To construct a mutant pEGFP- hTERTexpression vector, to observe its steady expression intransfected human bladder carcinoma cell line T24 and its role in molecular regulatory mechanisms of telomerase, and to provide a new target gene for bladder cancer. Methods: PCR amplification was performed by using primers basedon the known gene sequence of hTERT. PCR productionwas cloned into plasmid pGEMT-T easy and the sequenceof mutant hTERT gene was analyzed. A recombinantmutant hTERT vector (pEGFP-hTERT) was constructed at the EcoR I and Sal I sites of the pEGFP-C1 vector. Aftertransfecting the fusion gene into bladder carcinoma cell line T24 by calcium phosphate-DNA coprecipitation, the steady expression of GFP-hTERT fusion protein was tested by fluorescent light microscopy. The proliferation changes ofbladder carcinoma cell line T24 were detected by lightmicroscopy and senescence correlated b-galactosidase staining. Results: Identification of pEGFP-hTERT byenzyme digestion showed that mutant hTERT fragment had been cloned into EcoR I and Sal I sites of the pEGFP-C1 vector. The steady expression of GFP-hTERT fusion protein was localized in the nucleus of transfected cells. Expression of senescence-associated b-galactosidase in transfected cells gradually increased with extended cultured time and cellgrowth was suppressed. Conclusion: The mutant-type hTERT gene suppresses the proliferation of bladder carcinoma cell line T24 by competitive effect on telomerase activity. This suggests that hTERT gene might be a suitable gene target for bladder cancer therapy.

肝衰竭患者外周血单个核细胞hTERT mRNA及血清Ang-2和ICAM-1水平临床意义探讨

目的初步探讨肝衰竭患者外周血单个核细胞人瑞粒酶逆转录酶(hTERT)mRNA及血清血管生成素-2(Ang-2)和细胞间黏附分子-1(ICAM-1)水平变化的临床意义。方法2019年1月~2021年5月我院诊治的74例肝衰竭患者【亚急性肝衰竭(SALF)13例,慢加急性肝衰竭(ACLF)39例和慢性肝衰竭(CLF)22例】和同期健康体检者30例,分离并检测外周血单个核细胞(PBMC)hTERT mRNA水平,采用ELISA法检测血清Ang-2和ICAM-1水平。结果肝衰竭组PBMC hTERT mRNA、血清Ang-2和ICAM-1水平分别为(0.9±0.3)、(1344.6±55.3)ng/L和(540.2±22.4)ng/ml,显著高于健康人组【分别为(0.4±0.1)、(1062.5±36.2)ng/L和(167.3±15.6)ng/ml,P<0.05】;SALF患者PBMC hTERT mRNA、血清Ang-2和ICAM-1水平分别为(1.5±0.6)、(1507.8±38.2)ng/L和(647.3±26.2)ng/ml,显著高于ACLF患者【分别为(1.2±0.4)、(1398.6±35.8)ng/L和(582.7±24.1)ng/ml,P<0.05】或CLF患者【分别为(0.7±0.2)、(1280.5±46.3)ng/L和(468.9±20.3)ng/m,P<0.05】;经过1~3 m治疗,SALF组死亡3例,ACLF组死亡5例,CLF组死亡4例。结论肝衰竭患者PBMCs hTERT mRNA及血清Ang-2和ICAM-1水平显著升高,它们可能为判断肝损伤程度提供客观依据,值得进一步研究。