正常人白细胞与急性白血病细胞端粒酶分析

目的 探讨端粒酶活性检测在白血病临床病情分析中的价值。方法 采用改进的端粒重复扩增法分析正常人白细胞和急性白血病细胞的端粒酶活性。结果 急性白血病细胞表现出很高的端粒酶活性，并与白血病病情及异常染色体核型密切相关，而正常细胞活性很低或无，结论分析与白血病细胞异常端粒酶活性，对白血病诊断和临床治疗具有一定意义。     

急性白血病端粒酶检测及临床相关分析

目的　检测急性白血病细胞端粒酶活性与临床相关分析。方法　端粒重复扩增法 (TRAP)结合光密度定量分析端粒酶活性和临床病例资料分析。结果　在急性白血病初诊期或复发期均表现很高的端粒酶活性 ,缓解期病例活性降低 ,骨髓增生异常综合征转变为急性白血病者及慢性粒细胞白血病急性变后端粒酶活性明显增高。结论　急性白血病较高的端粒酶活性与临床病情演变相关 ,分析白血病细胞端粒酶活性 ,对白血病临床诊断和治疗具有一定意义。     

高白细胞性白血病患者细胞分离治疗及端粒酶检测

目的 :分析高白细胞性白血病细胞分离术治疗及端粒酶检测的临床价值。方法 :用血细胞分离机 ,对12例高白细胞性急性白血病患者进行白细胞分离治疗 ,并用端粒重复扩增法检测分离细胞和骨髓细胞端粒酶活性。结果 :经白细胞分离术后 ,10 / 12例患者临床症状减轻 ,白细胞计数降低至 5 0× 10 9/ L 以下 ,得以继续顺利化疗 ;患者伴有较高的端粒酶活性 ,其中 6 / 10例临床缓解时仍较高 ,且化疗治疗效果差。结论 :白细胞分离术可有效减轻白血病的细胞负荷 ,避免肿瘤细胞溶解综合征 ;高白细胞性白血病患者伴有较高端粒酶活性的生物特征需进一步研究。     

儿童急性白血病端粒酶活性检测及临床意义

目的：探讨端粒酶活性在儿童急性白血病中的表达及临床意义。方法：采用端粒重复系列扩增法（TRAP）检测了11例儿童急性白血病化疗前、完全解后骨髓单个核细胞（MNCs)端粒酶活性水平,对部分病例进行追踪观察,并与10例正常儿童骨髓MNCs端粒酶活性相比较。结果：正常骨髓MNCs具低水平的端粒酶活性,相对端粒酶活性范围2．6％－19．1％;急性白血病患儿化疗前相对端粒酶活性明显增高40．4％－98％,完全缓解后其相对端粒酶活性为21．7％－60．1％,随缓解时间的延长,端粒酶活性逐渐降低。结论：端粒酶激活在儿童急性白血病的发生中可能具有重要作用,动态观察端粒酶活性水平,对估测病情、监测微小残留病可能具有实际意义。     

白血病端粒酶活动度与三氧化二砷治疗观察

目的观察白血病端粒酶活性与三氧化二砷的治疗效果。方法端粒重复扩增法结合光密度测定和临床治疗观察。结果急性白血病均表现出较高的端粒酶活性，用三氧化二砷治疗后，获得临床血液学缓解及端粒酶活性降低，并在维持缓解间保持较低水平。结论观察端粒酶活动度有助于l临床判断病情转归及三氧化二砷的治疗反应。     

白血病细胞端粒酶活性的研究

目的：研究白血病细胞端粒酶活性表达及其意义。方法：采用PCR-ELISA半定量方法测定端粒酶活性。结果：68.6％(59／86)急性白血病(AL)患者和80％(12／15)慢性粒细胞白血病急性变(CML-BP)患者表达高端粒酶活性；而慢性粒细胞白血病慢性期(CML-CP)患者，无一例表达高端粒酶活性。急性淋巴细胞白血病患者端粒酶活性高于急性髓性白血病患者，CML-BP端粒酶活性高于CML-CP，而CML-CP端粒酶活性则低于正常对照组。AL端粒酶活性水平与患者的性别、年龄无关，也与治疗后缓解率无关。结论：激活或上调端粒酶活性在大多数AL发生和慢性粒细胞白血病急性变过程中起着重要作用；端粒酶活性测定可鉴别CML-BP与CML-CP；端粒酶可能是一潜在的治疗白血病的新靶点。     

淫羊藿甙对急性早幼粒白血病细胞端粒酶活性的影响

目的：探讨淫羊藿甙（ICA）对急性早幼粒白血病细胞端粒酶活性的影响。方法：采用ICA与全反式维甲酸（AT-RA）对比试验。观察ICA对白血病细胞端粒酶活性及增殖分化的影响。结果：ICA可明显抑制端粒酶活性，对白血病细胞均有较明显的诱导分化和抑制增殖作用。且与ATRA合用可产生明显的协同效应。结论：ICA具有抑制急性早幼粒白血病细胞端粒酶活性的作用。     

急性白血病端粒酶活性检测及其临床意义

目的：研究急性白血病发生发展不同阶段的端粒酶活性水平，探讨端粒酶活性检测对急性白血病的临床意义。方法：选取初治、治疗中和完全缓解期共33例成人急性白血病病例，以缺铁性贫血（IDA）和骨髓形态学正常的非血液病患者作对照，采用改良TRAP－银染法及亚利恩凝胶成像系统分析检测其骨髓标本内的端粒酶活性水平并随访。结果：缺铁性贫血患者端粒酶阴性或低水平表达；急性白血病患者端粒酶活性明显增高（P＜0．05），阳性率达70％，白血病完全缓解后端粒酶活性水平下降（P＜0．05），完全缓解期端粒酶活性仍高于正常者往往预后不良。结论：端粒酶与急性白血病的发生发展密切相关。端粒酶活性的检测可以作为白血病诊断及病情监测的一个生物学指标，有希望成为一种监测微小残留病的新的生物学标记。对白血病的抗端粒酶疗法有潜在的价值。     

硫醇酊对白血病细胞端粒酶逆转录酶表达和端粒酶活性选择性抑制的作用

为了面容硫醇类低分子化合物梳醇酊(Amifostine,AEF)对正常和恶性造血细胞中端粒酶,逆转录酶(hTERT),端粒酶活性的影响,多聚酶链反应(PCR)及酶联免疫吸咐方法用于检测白血病细胞及正常人单个核细胞hTERT mPNA表达及端粒酶活性.结果显示AMF可抑制白血病HL60细胞株以及急性白血病病人白血病细胞的hTERT转录表达,并进而导致端粒酶活性减低.正常活化淋巴细胞hTERT和端粒酶活性水平不受AMF影响.表明AMF能够选择性抑制肿瘤细胞端粒酶活性.     

Amplification of hTERT and hTERC genes in leukemic cells with high expression and activity of telomerase

Reactivation of telomerase plays an important role in carcinogenesis. Malignant cells almost always possess high activity and expression of telomerase. The aim of this study was to see whether there is any relationship between telomerase activity and expression and hTERT and hTERC gene amplification in acute lymphoblastic leukemia (ALL) and non-lymphoblastic leukemia (ANLL) cells. In addition telomere length was tested in leukemic cells at the time of diagnosis and during remission. Expression of the three components of telomerase (hTERT, hTERC and TP1) as well as telomerase activity was found both in ALL and ANLL cells. Telomerase activity was diminished in patients in remission. The leukemic cells showed considerable heterogeneity of terminal restriction fragments, that is telomere length. ALL cells showed a variable pattern of telomere length in contrast to ANLL cells which produced a predominantly short telomere pattern. Telomere length in the lymphocytes of leukemia patients was shorter in remission as compared to the time of diagnosis. FISH analysis revealed amplification of hTERT and hTERC genes in ALL and ANLL cells. Quantitative analysis showed that leukemic cells possess higher number of hTERT and hTERC copies than the normal PBL. Our results suggest that the activation of telomerase in leukemic cells is connected with amplification of hTERT and hTERC genes. The high expression and activity of telomerase found in leukemic cells may be partially explained by amplified hTERT and hTERC genes. Amplification of the telomerase genes seems to be a common event in carcinogenesis and may play a role in telomerase reactivation leading to cell immortalization.     

Cyclic adenosine 3':5'-monophosphate-dependent and -independent protein kinases in human leukemic cells

The pattern of protein kinase activity in leukemic cells from patients with chronic myelocytic leukemia, acute myeloblastic leukemia, acute monocytic leukemia, chronic lymphocytic leukemia, and acute lymphoblastic leukemia was studied and compared with normal peripheral blood granulocytes and lymphocytes. Our data showed that: (a) histone kinase activity was slightly lower in leukemic cells than in normal cells, whereas casein kinase activity was 2- to 3-fold higher in leukemic cells; (b) cyclic adenosine 3':5'-monophosphate stimulated 1.4- to 1.6-fold histone kinase activity of both normal and leukemic cells, whereas it did not stimulate casein kinase activity; (c) the ratio of histone kinase activities to casein kinase activities correlated directly with the maturation of the white blood cells; and (d) histone and casein kinase activities of extracts from normal and leukemic cells behaved similarly on chromatography on phosphocellulose and casein/Sepharose 4B. These results suggest that the increase in casein kinase activity is not due to the appearance of a new type of casein kinase but to an increase of the casein kinases 1 and 2 present in normal cells.     

Telomerase activity and telomere length in acute and chronic leukemia, pre- and post-ex vivo culture

We studied telomerase regulation and telomere length in hematopoietic progenitor cells from peripheral blood and bone marrow from patients with acute and chronic leukemia and myeloproliferative diseases. CD34+ cells from a total of 93 patients with either acute myeloid leukemia (AML; n = 25), chronic myeloid leukemia (CML; n = 21), chronic lymphocytic leukemia (CLL; n = 18), polycythemia vera (PV; n = 16), or myelodysplastic syndromes (MDS; n = 13) were analyzed before and in 19 patients after ex vivo expansion in the presence of multiple cytokines (kit ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor plus erythropoietin). Compared with hematopoietic progenitor cells from normal donors (n = 108), telomerase activity (TA) was increased 2- to 5-fold in chronic phase (CP)-CML, CLL, PV, and MDS. In AML, accelerated phase (AP) and blastic phase (BP)-CML, basal TA was 10- to 50-fold higher than normal. TA of CP-CML CD34+ cells was up-regulated within 72 h of ex vivo culture, peaked after 1 week, and decreased below detection after 2 weeks. In contrast, TA in AP/BP-CML and AML CD34+ cells was down-regulated after 1 week of culture and decreased further thereafter. The expansion potential of CD34+ cells from patients with leukemia was considerably decreased compared with CD34+ cells from normal donors. The average expansion of cells from leukemic individuals was 6.5-, 2.3-, 0.6-, and 0.2-fold in weeks 1, 2, 3, and 4, respectively, whereas expansion of normal cells was 5- to 15-fold higher. In serial expansion culture, a median telomeric loss of 0.7 kbp was observed during 3-4 weeks of expansion. Our results demonstrate that up-regulation of telomerase is similar in CD34+ cells from CP-CML, CLL, PV, and MDS patients and in normal hematopoietic cells during the first week of culture, whereas in AML and AP/BP-CML, telomerase is high at baseline and down-regulated during expansion culture. High levels of telomerase in leukemic progenitors at baseline may be a feature of both the malignant phenotype and rapid cycling. Telomerase down-regulation during culture of leukemic cells may be due to the decreased expansion potential or repression of normal hematopoiesis, or in AML it may be due to the partial differentiation of AML cells, shown previously to be associated with loss of TA. Telomere shortening during ex vivo expansion correlated with low levels of TA, particularly in chronic leukemic and MDS progenitors where telomerase was insufficient to protect against telomere bp loss during intense proliferation.     

Ectopic expression of c-myc fails to overcome downregulation of telomerase activity induced by herbimycin A, but ectopic hTERT expression overcomes it.

Telomerase plays a key role in the maintenance of chromosomal stability in tumors, but the mechanism regulating telomerase activity is still unclear. Recent studies have suggested that c-myc may be vital for regulation of hTERT mRNA expression and telomerase activity. In this study, we investigated the changes of telomerase activity and telomerase-related genes induced by herbimycin A in K562 human chronic myelogeous leukemic cells. Telomerase activity showed a biphasic pattern in herbimycin A-treated K562 cells. Initially, the telomerase activity decreased along with the decline of cells in S and G2/M phases, but it recovered slightly at the end of treatment. Expression of mRNA for the telomerase catalytic subunit (hTERT) was decreased before the decline of telomerase activity, and increased slightly before the reactivation of telomerase activity. During herbimycin A treatment, both c-myc and cyclin D1 mRNA showed transient downregulation before the increase of G1 cells. Herbimycin A treatment caused the downregulation of both telomerase activity and hTERT mRNA in cyclin D1-transfected K562 cells, while telomerase activity was partially restored in c-Myc-transfected cells. In contrast, hTERT-transfected K562 cells maintained a high level of telomerase activity during herbimycin A treatment. Neither the template RNA component of telomerase (hTERC) nor telomerase-associated protein (TEP-1) were altered in any of the transfected K562 cells. These results indicate that telomerase activity is mainly regulated by hTERT, and that c-Myc protein is one of the positive regulators of hTERT in leukemic cells but is not enough to counteract the downregulation of telomerase activity by herbimycin A completely.     

以hTERT基因反义核酸抑制端粒酶活性增加白血病细胞对CDDP诱导凋亡的敏感性

目的 以hTERT反义核酸抑制白血病细胞(HL-60和K562)端粒酶活性,研究CDDP诱导凋亡敏感性的变化。方法 全硫代反义核酸由上海生物化学研究所合成和纯化;端粒酶活性用试剂合测定(宝灵曼公司产品);用形态学方法和流式细胞仪检测细胞调亡。结果实验结果显示,hTERT全硫代反义核酸,通过下调hTERT基因表达,显著地抑制端粒酶活性;端粒酶活性下降以后,白血病细胞对CDDP诱导调亡的敏感性显著升高。结论 以hTERT基因反义核酸抑制端粒酶活性增加白血病细胞对CDDP诱导凋亡的敏感性。     

卵巢癌患者外周血癌细胞端粒酶活性检测的建立

目的　通过检测外周血中卵巢癌细胞的端粒酶活性 ,了解其血循环转移情况及其诊疗价值。方法　用改进的银染 -TRAP(端粒重复序列扩增 )法检测 3 3例卵巢癌患者术前及术后接受化疗后的外周血端粒酶活性 ,并以 10例卵巢良病变患者外周血作为对照。结果　 3 3例卵巢癌患者术前外周血端粒酶活性强阳性 8例 ,弱阳性 12例 ,术后接受化疗后仅 6例弱阳性 ;10例卵巢良病变患者外周血 1例端粒酶弱阳性 ,其余全部阴性。结论　卵巢癌患者外周血端粒酶活性检测在卵巢癌的治疗、预后的评估、随访等方面具有重要的临床意义。     

Telomere dynamics in myelodysplastic syndromes and acute leukemic transformation.

Myelodysplastic syndromes (MDS) are characterized by cytopenias in the blood and dysplastic features in the hematopoietic cells. Although the impact of cytogenetic abnormalities is considerable for prognosis, the exact genetic mechanism of MDS remains undetermined. In this study we assessed cytogenetic changes, microsatellite alterations, and telomere dynamics in order to obtain further insight into the pathogenesis of MDS. Thirty-three percentage of MDS patients and 60% of post-MDS acute leukemia (post-MDS AML) had de novo microsatellite changes. In the MDS phase, however, > 60% of patients showed reduction of telomere lengths without microsatellite changes, indicating that telomere reduction in most MDS patients does not seem to be directly linked to genome instability, or that reduction of telomere length does not induce microsatellite changes in the MDS phase. Some MDS patients had microsatellite changes without telomerase elevation, indicating that genome instability might accumulate during the disease progression in some MDS patients, and this condition (cellular senescence) may be related to ineffective hemopoiesis in MDS patients. In contrast, 40% of post-MDS AML patients had elevated telomerase activity with microsatellite changes, indicating that approximately 40% of patients with post-MDS AML patients had accumulation of genome instability resulting in elevated telomerase activity in an attempt to obtain genetic stability. However, the remaining MDS patients had microsatellite changes without telomerase up-regulation, suggesting that some MDS had genome instability even after leukemic transformation. Most MDS patients with elevated telomerase activity in the AML phase had elevated telomerase activity even in the MDS phase without apparent change in telomere length before and after leukemic transformation. These findings indicate that telomerase activity in the MDS phase may be independent of telomere length, although telomere shortening seems to be related to genomic instability, and this process may be linked to apoptosis of MDS cells.