Department of Pathology, Peking University Health Science Center, Beijing, China.
Pathol Int. 2010 May;60(5):386-94. doi: 10.1111/j.1440-1827.2010.02529.x.
Telomerase activity is found in various cell types including stem cells, neoplastic cells, and immortalized cells, suggesting a close association with their proliferation capacity. The telomeric repeat amplification protocol (TRAP) has been traditionally used to detect semi-quantitatively the telomerase activity by polyacrylamide gel electrophoresis (PAGE), which is difficult to apply for large scale analysis because of laborious post-PCR manipulation and potential carryover contamination. In the present study, a specific reverse primer was designed and the TRAP protocol was adapted to either PAGE or real-time PCR assay. Using cultured cell lines, the real-time TRAP showed a dramatic improvement in the reliability and accuracy of quantitation of telomerase activity and was able to discriminate the A549 cells from hundreds-fold human embryonic lung cells. Using clinical samples of 60 lung cancers and 8 inflammatory lesions, the real-time TRAP was also superior in quantitation, high-throughput capability and standardization. Our modified real-time TRAP should be applicable for the detection of telomerase activity for the initial screening and progression monitoring of lung cancer patients. Our approach is particularly useful when only limited clinical specimen is available, such as fine needle aspiration or other cytological specimens that may contain only a small number of tumor cells.
端粒酶活性存在于多种细胞类型中，包括干细胞、肿瘤细胞和永生化细胞，这表明其与细胞的增殖能力密切相关。传统上，端粒重复扩增协议（TRAP）通过聚丙烯酰胺凝胶电泳（PAGE）来半定量检测端粒酶活性，但由于 PCR 后处理操作繁琐且存在潜在的交叉污染，因此难以用于大规模分析。在本研究中，设计了一个特异性的反向引物，并对 TRAP 协议进行了调整，使其适用于 PAGE 或实时 PCR 分析。使用培养的细胞系，实时 TRAP 显著提高了端粒酶活性定量的可靠性和准确性，能够区分 A549 细胞和数百倍于人胚肺细胞。使用 60 例肺癌和 8 例炎症病变的临床样本，实时 TRAP 在定量、高通量能力和标准化方面也具有优势。我们改进的实时 TRAP 可用于检测端粒酶活性，用于肺癌患者的初步筛选和进展监测。当只有有限的临床标本可用时，如细针抽吸或其他可能只含有少量肿瘤细胞的细胞学标本，我们的方法特别有用。
