SELDI-TOF-MS联合LCM技术筛选大肠癌肝转移早期诊断标志蛋白

Application of SELDI-TOF-MS and LCM to screen protein markers for early diagnosis of liver metastasis of colorectal carcinoma

目的:应用表面增强激光解吸电离飞行时间质谱蛋白质芯片(SELDI-TOF-MS)联合激光显微切割(LCM)技术筛选大肠癌及其肝转移标志蛋白.方法:采用LCM技术获取24例大肠癌肝转移患者正常大肠、原发灶及肝转移灶癌细胞;应用SELDI-TOF-MS技术对其行蛋白质谱分析;采用Biomarker Wizard软件分析差异蛋白;通过查询蛋白库对特定分子质量所对应的标志蛋白进行初步确定.结果:比较3组细胞间的SELDI质谱图,发现大肠癌原发灶与正常大肠两组间存在15个标志蛋白,12个表达上调,3个表达下调;大肠癌肝转移灶与原发灶两组间存在9个标志蛋白,5个表达上调,4个表达下调.其中质荷比4676.63Da,11740.87Da,21063.59Da和22783.36Da,蛋白峰差异性最明显(P<0.01).通过查询ExPasy蛋白库筛选出20个差异蛋白,包括整合膜蛋白2C、DNA修复蛋白RAD51同系物4、细胞周期检查点蛋白RAD1、人附睾蛋白4、着丝粒蛋白R、Pleckstrin同源结构域家族成员3等.其中细胞凋亡调节Bax蛋白γ亚型、蛋白质S100A11(Protein S100-A11)、Raf激酶抑制蛋白(RKIP)和热休克蛋白27(HSP-27)在正常大肠、原发灶及肝转移灶癌细胞中均呈差异性表达,并且差异性最明显(P<0.01).结论:SELDI蛋白质芯片联合LCM技术有可能筛选出敏感性高、特异性强的大肠癌标志蛋白,筛选出的差异蛋白可能是大肠癌及其肝转移特异性生物标志物.

AIM：To screen protein markers for liver metastases of colorectal cancer using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry protein chip（SELDI-TOF-MS） in combination with laser capture microdissection（LCM） technology.METHODS：Normal colorectal cells,primary tumor cells and liver metastatic cells were obtained from 24 colorectal cancer patients with liver metastases using the LCM technology.Protein prof iling was performed using SELDI-TOFMS technology.Differentially expressed proteins were analyzed using Biomarker Wizard soft-ware and identif ied by querying the database.RESULTS：Fifteen differentially expressed proteins were found between primary tumor cells and normal colorectal cells,of which 12 were up-regulated and 3 down-regulated.Nine differentially expressed proteins were found between liver metastatic cells and primary tumor cells,of which 5 were up-regulated and 4 downregulated.Twenty differentially expressed proteins were identified by querying ExPasy protein database,including integral membrane protein 2C,DNA repair protein RAD51 homolog 4,cell cycle checkpoint protein RAD1,human epididymis protein 4,centromere protein R,and pleckstrin homology domain family member 3.Apoptosis regulator Bax-γ,S100A11,Raf kinase inhibitor protein（RKIP） and heat shock protein 27（HSP-27） displayed most obvious differential expression among normal colorectal cells,primary tumor cells and liver metastatic cells（all P 0.01）.CONCLUSION：SELDI-TOF-MS technology combined with LCM may allow to screen highly sensitive and specif ic protein markers for colorectal cancer and liver metastases of colorectal cancer.