GSTP1在先天性巨结肠症中蛋白质表达谱、甲基化检测与表达分析

Analysis of Protein Profile and Expression and Detection of GSTP1 Methylation in Hirschsprung Disease of Tissues

目的 利用双向凝胶电泳-基质辅助激光解吸离子化飞行时间质谱（matrix-assisted laser desorption ionization time of flight mass spectrometry,MALDI-TOF-MS）技术进行先天性巨结肠症（hirschsprung disease,HD）中蛋白质表达谱的研究;寻找HD中的生物标志物,为临床HD的早期无创性诊断提供依据.方法 分别提取20例HD患者配对狭窄段及正常段肠管组织的总蛋白质,进行双向凝胶电泳、银染,两组凝胶图像差异分析,得到差异蛋白质.对目的谷胱苷肽-S-转移酶1（glutathione S-transferase pi,GSTP1）蛋白质进行MALDI-TOF-MS质谱分析.运用甲基化特异性聚合酶链反应方法检测HD中GSTP1基因启动子区5′端 CpG 岛甲基化程度与表达.采用荧光实时定量PCR方法检测HD中GSTP1基因的mRNA 转录水平.结果 获得了分辨率较高的HD狭窄段及正常段肠管组织双向凝胶电泳图谱;得到阳性结果的差异蛋白质谷胱甘肽-S-转移酶（glutathione S-transferase,GST）.MSP 检测48 例HD,其中狭窄段肠管组织GSTP1基因高甲基化41例;而正常段肠管组织GSTP1基因甲基化仅7例.HD狭窄段肠管组织GSTP1基因mRNA表达低于正常段肠管组织（P〈0.05）.结论 利用双向凝胶电泳-MALDI-TOF-MS质谱技术检测HD组织差异蛋白质所获得的GSTP1蛋白可为寻找HD早期诊断的分子标记物提供理论依据;GSTP1基因异常甲基化、表达与HD的发生发展相关.

Objective This study was aimed to study hirschsprung disease （HD） protein profile in tissues by two-dimensional gel eleetrophoresis （2-DE） -matrix-assisted laser desorption ioni- zation time of flight mass spectrometry （MALDI-TOF-MS） , explore HD biomarkers, and provide theoretical basis for early non-invasive diagnosis in clinic. Methods Total proteins were extracted from HD tissues of 20 couples of stegnotic versus normal segments 2DE and silver staining were performed to analyze the differences in get image between the two groups. Obtained glutathione S-transferase pi （GSTP1） proteins were subject to MALDI-TOF-MS mass spectrometry. Methylation degree of 5-CpG island in GSTP1 gene promoter region in HD tissues were detected by MSP. GSTP1 mRNA transcription levels in HD tissues were detected by quantitative real-time PCR （qRT-PCR）. Results We got a high-resolution 2DE of HD tissues. Positive glutathione S-transferase （GST） differential proteins were hypothetical proteins. The MSP test results demonstrated that 41 of 48 stegnotic segment cases presented GSTP1 gene hypermethylation, demonstrated that 7 of 48 normal segmen cases presented GSTP1 gene hypermethylation. The mRNA expression levels of GSTP1 gene were significantly lower in HD of stegnotic segment than that in normal segment （ P 〈 0.05 ）. Conclusions Utilization of 2DE-MALDI-TOF-MS mass spectrometry in detect of HD tissues is useful for identying differential proteins. The obtained results may facilitate further study of HD proteomics. Hypermethylation and expression of GSTP1 gene is correlated with development and progression of HD. Screened GSTPI Proteins may help to find out markers of early diagnosis of HD.