提出了一种检测三磷酸腺苷(ATP)的非标记荧光新方法,该方法基于T4多聚核苷酸激酶调控Lambda核酸外切酶活性的原理,利用DNA荧光染料SYBR Green I作信号报告基团.有ATP时,双链DNA底物能在T4 PNK作用下发生5'端磷酸化,进而被核酸外切...提出了一种检测三磷酸腺苷(ATP)的非标记荧光新方法,该方法基于T4多聚核苷酸激酶调控Lambda核酸外切酶活性的原理,利用DNA荧光染料SYBR Green I作信号报告基团.有ATP时,双链DNA底物能在T4 PNK作用下发生5'端磷酸化,进而被核酸外切酶降解成DNA碎片,得到较弱的荧光信号,信号强度与ATP浓度成反比.该方法对ATP检测的线性范围为0.04~4 mmol/L,检测限为20 nmol/L.实验结果表明本方法具有快速、成本低、灵敏度高和简单易操作等优点,可进一步应用于复杂生物样品的分析.展开更多
Objectives To explore the pathogenesis of rheumatoid arthritis (RA) by studying the expression of T cell receptors (TCRs).Methods T cell receptor Vβ (TCR Vβ) gene usage and expression were analyzed from synovial mem...Objectives To explore the pathogenesis of rheumatoid arthritis (RA) by studying the expression of T cell receptors (TCRs).Methods T cell receptor Vβ (TCR Vβ) gene usage and expression were analyzed from synovial membrane and peripheral blood of 8 RA patients, 2 osteoarthritis patients and 2 accident amputees. The complementary determining region 3 (CDR3) of 25 TCR Vβ subfamily genes in unselected T cell populations were amplified semi-quantitatively by reverse transcription-polymerase chain reaction (RT-PCR). The products were further studied by genescan for frequency of Vβ usage.Results The numbers of Vβ subfamilies expressed by T cells from RA peripheral blood and synovial membrane were not significantly restricted. More importantly, biased Vβ gene expression in RA synovium was observed and Vβ6, Vβ17, and Vβ22 genes were the predominant subfamilies. It was noteworthy that the expression of Vβ17 in RA synovium was significantly increased. Conclusion Our data were consistent with the hypothesis that several antigen or superantigen-driven processes may be involved in the pathogenesis of RA.展开更多
文摘Objectives To explore the pathogenesis of rheumatoid arthritis (RA) by studying the expression of T cell receptors (TCRs).Methods T cell receptor Vβ (TCR Vβ) gene usage and expression were analyzed from synovial membrane and peripheral blood of 8 RA patients, 2 osteoarthritis patients and 2 accident amputees. The complementary determining region 3 (CDR3) of 25 TCR Vβ subfamily genes in unselected T cell populations were amplified semi-quantitatively by reverse transcription-polymerase chain reaction (RT-PCR). The products were further studied by genescan for frequency of Vβ usage.Results The numbers of Vβ subfamilies expressed by T cells from RA peripheral blood and synovial membrane were not significantly restricted. More importantly, biased Vβ gene expression in RA synovium was observed and Vβ6, Vβ17, and Vβ22 genes were the predominant subfamilies. It was noteworthy that the expression of Vβ17 in RA synovium was significantly increased. Conclusion Our data were consistent with the hypothesis that several antigen or superantigen-driven processes may be involved in the pathogenesis of RA.
