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目的 鉴别并获取与 K562细胞向红系分化相关的表达序列标签 (expressed sequence tags,EST)。方法利用改进的差异显示反转录聚合酶链反应 (differential display reverse transcription polym erase chain reaction DDRT-PCR )方法,分析诱导前及用氯高铁血红素 (hem in)诱导 36h 后 K 562细胞的差异表达 cDNA 片段,挑选其中差异明显的cDNA 片段,经克隆、测序和基因信息学分析,挑选代表新序列或有一定功能线索的差异表达 EST 进行 Northern 印迹分析。结果 获得诱导前后 K 562细胞的差异表达 cDNA 片段 60条,其中 38条表达上调,22条表达下调。对 40个差异表达明显的 cDNA 片段的生物信息学分析说明,23个 EST 与 GenBank 中已知序列有 95%以上同源性,10个为只与 dbEST 有高度同源的序列,7个未发现同源序列;进行 Northern 印迹分析的 8个差异表达 EST 中,6个 EST 的杂交结果与 DDRT-PCR 显示的差异表达状况一致。结论 改进的 DDRT-PCR 方法能够较好地克服假阳性问题,这些差异表达 EST 的获得为进一步研究红系分化的分子机制和控调网络提供了一定线索。
Objective To identify and obtain expressed sequence tags (ESTs) related to erythroid differentiation of K562 cells. Methods Differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used to analyze the differential expression of K 562 cells before induction and after 36 h induction with hemin The cDNA fragments were selected and the cDNA fragments with obvious difference were selected. Northern blotting analysis was carried out by cloning, sequencing and geneticinformation analysis. Results There were 60 cDNA fragments differentially expressed in K 562 cells before and after induction, of which 38 were up-regulated and 22 were down-regulated. Bioinformatic analysis of 40 differentially expressed cDNA fragments revealed that 23 ESTs shared 95% homology with known sequences in GenBank, 10 were highly homologous to dbEST, and 7 were not found Homology; Northern blot analysis of 8 differentially expressed ESTs, 6 EST hybridization results and DDRT-PCR showed the same differentially expressed status. Conclusion The improved DDRT-PCR method can better overcome the false positive problem. The availability of these differentially expressed ESTs provides some clues for further studying the molecular mechanisms and regulatory networks of erythroid differentiation.