应用限制性显示技术筛选K562细胞脱核前后差异表达基因

Screening differentially expressed genes in denucleated K562 cells with restriction display technique

目的应用限制性显示(RD)PCR技术筛选细胞松弛素B(Cytochalasin B,CB)诱导K562细胞脱核前后的差异基因,探讨CB诱导细胞脱核的分子机制。方法用CB(终浓度为10 μg／ml)处理K562细胞24 h,分别提取对照组和处理组细胞总RNA,将两组等量的细胞总RNA纯化为mRNA,反转录为cDNA,利用RD-PCR技术和聚丙烯酰胺凝胶电泳分离、筛选CB处理前后差异表达的基因。对筛选出的差异表达基因进行克隆、测序,在GenBank检索进行序列同源性分析。结果筛选及克隆了7个差异表达基因,其中水通道蛋白1(AQP1)基因经反转录PCR验证在CB诱导脱核的 K562细胞中表达上调。结论 AQP 1基因在CB诱导K562细胞脱核过程中特异性高表达,提示其可能与细胞脱核及增殖抑制密切相关。

Objective To screen differentially expressed genes in cytochalasin B （CB）-induced denucleated K562 cells by restriction display （RD） technique. Methods The total RNA was isolated and purified from K562 cells before and after CB （10 μg/ml） treatment. The mRNA from both treated and untreated K562 cells were reversly transcribed into cDNA, and the differentially expressed genes were separated using RD technique combined with polyacrylamide gel electrophoresis and sliver staining, followed by cloning, sequencing and homology analysis against GenBank database of these genes. Results Seven differentially expressed genes were identified in CB-treated cells including aquaporin 1 （AQP1） gene, which was verified to be up-regulated after CB treatment by RT-PCR. Conclusion AQP1 gene might be in close association with the regulation of denucleation processes and CB-induced proliferation inhibition ofK562 cells.