【摘要】 目的检测胃癌细胞中SHP1基因启动子区CpG岛异常甲基化状态。方法使用甲基化分析软件Cpgplot预测SHP1基因转录起始位点-1 000 bp~1 340 bp区域的CpG岛并用MethPrimer软件设计甲基化特异性PCR(MSP)引物;建立一种基于Bisulfite修饰、无需提取DNA而直接检测少量细胞甲基化的新方法,并将其用于胃癌细胞SHP1基因启动子区甲基化状态的检测;克隆MSP产物、Sanger测序、分析测序峰图;比较Bisulfite修饰前后序列并分析CpG位点甲基化状态。结果 MSP引物均位于SHP1基因第一外显子区近转录起始位点;CpG岛长度>200 bp、GC含量>50%、Obs/Exp>0.60;MSP检测仅出现甲基化引物特异性扩增条带,提示该区域高甲基化。结论胃癌细胞中SHP1基因启动子区CpG岛存在高甲基化。更多还原

【Abstract】 Objective To investigate the aberrant DNA methylation status of CpG islands in SHP1 gene promoter region in gastric cancer cells. Method CpG islands in the region of-1 000 bp-1 340 bp in transcription start sites of SHP1 gene were analyzed by Cpgplot software, and the primers of methylationspecific PCR(MSP) were designed by MethPrimer software. A new bisulfite-based MSP method was applied to detect the methylation status of SHP1 gene promoter region the limited gastric cancer cells. Bisulfite-modified DNA samples were amplified by MSP primers covering the CpG islands in SHP1 gene promoter region. The MSP products were purified cloned, which was subject to Sanger sequencing. Original and bisulfite modified partial sequences were compared. CpG loci methylation status was analyzed. Results Cpgplot displayed that MSP primers were located in SHP1 gene exon1 area which was close to the transcription initiation site. CpG islands length was > 200 bp, GC content was > 50%, and Obs/Exp was > 0.60. MSP detection showed that methylation-specific primer amplification was positive, whereas the unmethylation-specific primer amplification was negative, which revealed the region was highly methylated. Conclusion CpG islands in SHP1 gene promoter region are hypermethylated in gastric cancer cells.更多还原