端粒重复序列扩增-银染色法用于端粒酶活性测定的研究

Detection of telomerase activity using telomere repeat amplification protocol silver stain

目的 :建立端粒重复序列扩增 (TRAP) -银染色技术 ,探讨端粒酶活性检测在肿瘤诊断中的意义。 方法 :用裂解液提取组织细胞中的端粒酶模板 ,在特异引物作用下进行 PCR扩增 ,所得产物用氯仿∶异戊醇 (2 4∶ 1)处理浓缩 ,作聚丙稀酰胺凝胶垂直板电泳 ,经硝酸银染色分析端粒酶活性。 结果 :TRAP-银染色法能准确特异地检测小鼠骨髓瘤细胞 (SP2 / 0 )的端粒酶活性 ,灵敏度可达 1× 10 2个细胞。端粒酶阳性率在 2 3例各种恶性肿瘤组织中为 86 .9% (2 0 / 2 3) ,而在 19例炎性包块与良性增生组织为 5 .3% (1/ 19) ,5例正常组织中未发现有端粒酶活性。 结论 :TRAP-银染色法简便快速、具有较好的测定敏感度与重复性 ,对临床恶性肿瘤疾病的诊断具有良好的应用前景。

Objectives:To establish a method of telomere repeat amplification protocol(TRAP)silver stain technique, and to investigate the significance of telomerase activity detection in tumor diagnosis. Methods:Telomerase template was extracted from tissues and cells by lysis buffer, PCR amplification was performed under specific primer elongation. After processed and enriched with chloroform∶isopentanol(24∶1),the PCR products were electrophoresed by vertical plate on 10% invariance polyacrylamide gel. Telomerase activity was analyzed using sliver nitrate nucleic acid gel stain. Results:Telomerase activity could be determined accurately and specifically by TRAP silver stain in mice myeloma cells, and the sensitivity of detection of this method was 1×10 2 cells. The positive rate of telomerase was 86.9%(20/23) of patients with various tumor tissues, and 5.3%(1/19) of patients with inflammation as well as benign proliferation tissues, while telomerase activity was not detected in 5 normal tissues. Conclusions:TRAP silver stain proctocol is simple, rapid, sensitive and reproducible. It is applicable in the diagnosis of clinical malignant diseases.