白血病细胞系来源的P2X7受体的功能研究

P2X7受体是ATP门控的离子通道。从白血病细胞系J6-1细胞中扩增P2X7受体编码区全序列,克隆到pTARGET真核表达载体,经DNA序列分析后转染Ramos细胞,获得稳定表达细胞株;应用RT-PCR、Westernblot和流式细胞术检测P2X7受体在Ramos中的表达;荧光分光光度计检测P2X7受体介导的胞内钙离子浓度变化。结果显示,J6-1细胞来源的P2X7受体在第559位有一个A→G的有义突变,导致Asn187→Asp187,可在Ramos细胞中表达,在特异性激动剂BzATP作用下可引起胞内钙离子浓度的升高,但所需激动剂浓度高于常规浓度。

Study on The Function of P2X7 Receptor from Leukemia Cell Line

P2X7 receptor is an ATP-gated cation channel. The entire coding sequence of P2X7 receptor from J6-1 human leukemia cells was cloned and inserted into pTARGET plasmid to construct eukaryotic expressing plasmid followed by DNA sequencing. Ramos cell clones stably expressing P2X7 receptor were obtained after transfection and G418 selection. RT-PCR, Western blot analysis and FACS were used to detect the expression of P2X7 receptor in these clones. The cytoplasmic free calcium concentration was monitored by fluorospectrophotometer. The results showed that the sequence of P2X7 receptor gene from J6-1 cells had a substitution of A559→G559, causing a substitution of Asn187→Asp187 in amino acid sequence. This P2X7 receptor can be expressed in Ramos cells and mediate rise of cytoplasmic free calcium concentration when stimulated by higher concentration of specific agonist, BzATP.