Development of novel bioluminescent assay for telomerase and its application to diagnostic of cancer

端粒是真核生物染色体末端的特殊结构。在人类染色体中，端粒由数千个拷贝的6个碱基重复序列（TTAGGG）组成。虽然人类体细胞通过减少端粒重复的细胞分裂诱导细胞死亡，但癌细胞通过端粒酶诱导端粒重复的延伸。端粒酶是一种核糖核蛋白，其使用其RNA组分作为模板合成并将端粒重复序列引导到现有端粒的3'端。因此，端粒酶参与细胞的恶性转化或永生化，并作为抗癌药物筛选和诊断肿瘤的标志物而受到关注。端粒重复序列扩增法（TRAP）是目前检测端粒酶的通用方法。然而，这些方法通常采用丙烯酰胺凝胶电泳扩增端粒重复聚合酶链反应（PCR）后，因此，TRAP方法需要相当长的时间和技能，我们。我 关于我们 为了快速、高灵敏地检测端粒酶活性，我们建立了一种新的端粒酶生物发光检测方法，即用丙酮酸磷酸二激酶（PPDK）转化端粒酶反应和PCR反应产生的焦磷酸，再用萤火虫荧光素酶反应检测ATP，其检测限为1.0×10- 15 mol/assay。为了优化端粒酶活性的生物发光检测方法，我们设计了端粒重复序列的特异性引物，并选择了高效的Ta q聚合酶进行PCR。PCR扩增端粒酶反应产物的正、反义引物序列分别为5 '-AATCCGTCGAGCAGAGTT-3'和5 '-CTAACCCTAACCCTAACC-3'。在Taq聚合酶的研究中，利用钛Taq DNA聚合酶可获得高效的PCR扩增。在端粒酶反应和随后的PCR之后，通过所提出的生物发光测定来检测所释放的焦磷酸盐。结果，阳性细胞（500个细胞）和非活性细胞（通过在85℃下加热10 min制备）可明确鉴别。然后，生物发光强度的时间过程进行了检查。最大生物发光强度维持约两分钟。用500、250、100、50、10、5、1个细胞检测端粒酶细胞的检测限。1个细胞/测定通过端粒酶反应30分钟和由33个循环组成的PCR检测。检测了PCR循环数，检测到25个循环。目前，我们正在研究更简单和快速的生物发光检测方法，并正在开发其在癌症临床化学和再生医学等基础研究中的应用。少

Telomeres are specific structures found at the end of chromosomes in eukaryotes. In human chromosomes, telomeres consist of thousands of copies of 6 base repeats (TTAGGG). Although human somatic cells induce cell-death by reduction of telomeric repeats with cell division, cancer cells induce extension of telomeric repeats by telomerase. Telomerase is a ribonucleoprotein that synthesizes and directs the telomeric repeats onto the 3’ end of existing telomeres using its RNA component as a template. Therefore, telomerase participates in malignant transformation or immortalization of a cell, and attracts attention as anticancer drug screening and diagnostic tumor marker. Recently, telomeric repeat amplification protocol (TRAP) is used as universal method of telomerase assay. However, these approaches generally employ acrylamide gel electrophoresis after amplifying telomeric repeat by polymerase chain reaction (PCR); as a result, the TRAP method requires considerable time and skill for us. I … More n this study, for rapid and high sensitive detection of telomerase activity, we developed novel telomerase assay using bioluminescent detection method; that is, pyrophosphates produced by telomerase reaction and PCR are converted ATP by pyruvate phosphate dikinase (PPDK), and ATP is detected by firefly luciferin-luciferase reaction.As a result, the detection limit of pyrophosphate was 1.0×10-15mol/assay. For optimal bioluminescent assay of telomerase activity, we designed the specific primers to the telomeric repeat and selected the efficient Ta q polymerase for PCR. Sequences of the sense and antisense primers for PCR amplification of telomerase reaction product were 5’-AATCCGTCGAGCAGAGTT-3’ and 5’-CTAACCCTAACCCTAACC-3’, respectively. In study of Taq polymerase, efficient PCR amplification could be obtained by use of TITANIUM Taq DNA polymerase. After the telomerase reaction and subsequent PCR, the released pyrophosphate was detected by the proposed bioluminescent assay. As a result, positive cell (500 cells) and inactive cell (prepared by heating at 85℃ for 10 min) could be clearly identified. Then, time course of bioluminescent intensity are examined. The maximum bioluminescence intensity was maintained for about two minutes. The detection limit of cells with telomerase was examined with 500, 250, 100, 50, 10, 5, 1 cell. 1 cell/assay was detectable by telomerase reaction for 30 minutes and PCR consisting of 33 cycles. PCR cycle number also was examined, 25 cycles was detectable. Presently, we are examining simpler and rapid bioluminescent detecting method, and we are developing its applying to clinical chemistry of cancer and to basic research such as regenerative medicine. Less