PCR-ELISA和PCR-PAGE法检测血液肿瘤细胞株的端粒酶活性  

Detecting telomerase activity in malignant hematological cells by PCR-ELISA and PCR-PAGE

作　　者：何冬梅[1] 张洹[1] 

机构地区：[1]暨南大学医学院血液病研究所,广东广州510632

出　　处：《暨南大学学报（自然科学与医学版）》2003年第2期42-45,共4页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：广州市科技计划重点基金资助项目(2001-Z-037-01)

摘　　要：　目的:通过端粒酶聚合酶链反应-酶联免疫测定(PCR-ELISA)和聚合酶链反应-聚丙烯酰胺凝胶电泳(PCR-PAGE)方法检测血液肿瘤细胞株HL-60、K562、Raji的端粒酶活性,探讨其临床应用价值。方法:PCR-ELISA方法是将端粒重复序列PCR变性产物与地高辛标记且对端粒重复片段特异的探针杂交,杂交产物通过ELISA进行测定。PCR-PAGE法是将端粒重复序列PCR产物直接进行聚丙烯酰胺凝胶电泳,通过硝酸银染色测定端粒酶活性。结果:在端粒酶PCR-ELISA方法检测中,PCR在25～35次循环之间,吸光度A值与循环次数呈线性关系,35～45次循环已达到平台期。PCR-ELISA法检测显示HL-60、K562、Raji细胞均表达高水平的端粒酶活性,吸光度A均值分别为2 362、2 336、2 145。对HL-60、K562、Raji细胞的端粒重复序列扩增的PCR产物直接进行PAGE电泳、银染均呈现3条以上间隔6bp的特征性阶梯状条带。结论:对端粒重复序列PCR产物可同时进行ELISA检测和PAGE-银染,两种方法均可用于恶性肿瘤的临床诊断,ELISA法更为灵敏而简单,且可相对定量。Aim: To detect telomerase activity in malignant hematological cells by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA) and polymerase chain reaction-polyacrylamide gel electrophoresis (PCR-PAGE), and to investigate their clinical signifcance. Methods: The telomerase PCR-ELISA is an assay in which telomeric repeat amplification products were detected by ELISA. The PCR-PAGE assay uses polyacrylamide gel electrophoresis to determine telomeric repeat amplification products. Results: A linear relationship between the absorbance and the number of PCR cycles was obtained within 25～35 cycles. A typical plateau effect reached after 35 PCR cycles. HL-60, K562, Raji cells were found to express high levels of telomerase activity by PCR-ELISA assay in which the absorbance were 2362, 2336, 2145, respectively. The telomeric repeat amplification products by PAGE-sliver staining showed a typical ladder with the telomerase-specific 6-nucleotide increments. WConclusion:ELISA and PAGE-sliver staining in the meantime can show telomeric repeat amplification products. Both of them could be suitable for clinical application, but PCR-ELISA assay is more simple and sensitive.

关 键 词：端粒酶 聚合酶链反应 酶联免疫测定 聚丙烯酰胺凝胶电泳 

分 类 号：R733.7[医药卫生—肿瘤]