白血病细胞中共表达P2X7受体和Notch1胞内区

Dual Over-expression of P2X7 Receptor and Intracellular Domain of Notch1 in Leukemia Cells

本研究旨在构建野生型及N187D突变型P2X7与ICN1基因的共表达载体,并在白血病细胞中表达,为进一步探讨P2X7在白血病发展中的作用奠定基础。采用Overlap PCR法,通过2A连接,构建野生型或N187DP2X7与ICN1的共表达载体;经DNA序列分析验证后,包装病毒、感染K562细胞并经细胞分选获得稳定感染细胞系。采用RT-PCR、Western blot、胞浆游离钙离子浓度测定等方法,验证外源基因的表达及P2X7受体的功能。结果表明,序列分析显示载体构建正确;包装病毒对K562细胞感染效率40%-70%,流式分选获得稳定表达细胞株;RT-PCR结果显示,P2X7受体和/或ICN1基因可在对应的稳定表达细胞株中高效表达;Western blot也证明,P2X7受体可在感染编码P2X7受体基因病毒的K562细胞中高效表达;胞浆游离钙离子浓度检测显示,在BzATP刺激下,表达野生及N187D突变型P2X7的K562细胞胞浆游离钙离子浓度持续升高。结论:本研究在白血病细胞中成功共同高表达P2X7受体与ICN1,为进一步探讨野生型和N187D突变型P2X7受体在白血病发展中的作用奠定基础。

This study aimed to construct the dual expression vectors of wide type or N187D mutant P2X7 receptor and intracellular domain of Notchl （ 1CN1 ） linked by 2A peptide to coexpress them in leukemia cells so as to lay a foun dation for further investigating the role of P2X7 in development of leukemia. Overlap PCR was used to construct the dual expression vectors encoding wide type or N187D mutant type P2X7 receptor and ICN1 linked by the self-cleaving 2A se quence. The results showed that stable expressing cell lines were obtained by retroviral infection followed by cell sorting after DNA sequence analysis. RT-PCR, Western blot, intracellular free calcium concentration analysis were used to veri- fy the functionally successful construction of K562 cell line expressing P2X7 receptor alone or with ICN1. DNA sequence analysis revealed that all construction were right. The infection efficiency of packaged constructed virus ranged from 40% to 70% for K562 cells. Stable infected cell line was obtained by cell sorting. RT-PCR analysis revealed that P2X7 receptor and/or ICN1 could be detected at high level in their stable infected cell lines, respectively. Western blot analysis also showed that P2X7 receptor was highly expressed in cell line infected by virus with P2X7 receptor. Sustained increase in intraceUular free calcium concentration （ E Ca2 ＋ ] i ） could be observed in K562 cells overexpressing either type of P2X7 receptor upon stimulation with BzATP. It is concluded that the wide type or N187D mutant P2X7 receptor and ICN1 are simultaneously and functionally over-express in leukemia cells, which lay a foundation for further studying the role of P2X7 receptor in the development of leukemia.