【摘要】 目的:建立端粒酶活性定量检测的TRAP-发光分析法。方法:细胞株、组织样品与引物孵浴后,释放的焦磷酸盐由硫酸化酶作用转变为ATP,用荧光素酶生物发光系统检测发光信号;比较TRAP-发光分析法与TRAP-ELISA的结果。结果:TRAP-发光分析法的线性范围在2～1000个细胞之间。检测结果与TRAP-SYBR Green染色一致,与TRAP-ELISA法显著相关(r2=0.992,P<0.001)。结论:TRAP-发光分析法是一种稳定、快速、实际可行的端粒酶活性的定量分析方法。更多还原

【Abstract】 Objective To establish a quantitative method to measure telomerase activity by bioluminescence connected with telomeric repeat amplification protocol(TRAP). Methods Telomerase activity was measured by evaluating the amount of pyrophosphate (PPi) generated in PCR amplification of teolmerase elongation product, with the use of the sensitive enzymatic luminometric inorganic pyrophosphate detection assay(ELIDA). Results TRAP connected with ELIDA (TRAP-ELIDA) could quantitatively detect telomerase activity within linearity from 2 to 1 000 cell equivalents. The ELIDA signals accorded with results of TRAP SYBR Green staining, and the results of ELIDA were significantly correlated to that of TRAP connected with enzyme-linked immunosorbent assay (TRAP-ELISA) (r2=0.992, P < 0.001). Conclusions TRAP-ELIDA is a simple and sensitive method to quantify telomerase activity without time-consuming gel electrophoresis. Because of the luminometer by which TRAP-ELIDA is used to measure telomerase activity,TRAP-ELIDA can be applied to a large number of clinical samples at the same time.更多还原