摘要
目的 筛选并鉴定小儿肾母细胞瘤血清创伤应激相关因子,提高肾母细胞瘤血清特异蛋白标志物的敏感性和准确度.方法 将样品分组进行SELDI-TOF-MS检测,筛选特异性差异蛋白质,然后用HPLC和MALDI-TOF-MS对差异血清蛋白进行纯化,酶解蛋白质样品后,LC-Ms/MS测定该蛋白酶解肽段,将所得PMF经Mascot检索程序查询连接蛋白质序列Swissprot数据库,进行数据库检索,并应用Western blotting对于以上筛选及鉴定的差异性蛋白质进行验证.结果 SELDI-TOF-MS检测筛选出m/z位于5 816的差异性蛋白质,在肾母细胞瘤组高表达（强度为2128.3＆#177;137.2）,正常小儿组明显低表达（强度为30.4＆#177;7.8）,差异有统计学意义（P＜0.01）,经交叉检测,在测试集上判别该标志物组合模型的特异性为100％,敏感度为100％.正常血清组和创伤应激血清组比较筛选到差异最显著的m/z位于5 816的蛋白质标志物,在创伤组血清中高表达（强度为3674.6＆#177;255.7）,正常组中呈明显低表达（强度为30.4＆#177;7.8）,差异有统计学意义（P＜0.01）.经鉴定及验证此差异性蛋白质为硫氧还蛋白1 （thioredoxin Trx1）.结论 小儿肾母细胞瘤血清中含创伤应激相关炎症因子：硫氧还原蛋白1 （thioredoxin Trx1）.
Objective To screen and identify the serum traumatic stress related protein markers of pediatric nephroblastoma so as to improve their detection sensitivity and accuracy.Methods Three groups of samples were detected by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry （SELDI-TOF-MS） to screen for proteins of specificity differences.Then high-performance liquid chromatography （HPLC） and MALDI-TOF-MS were performed for purifying discrepant serum proteins.Enzymatic digestion was used to decompose protein samples.Liquid chromatographymass spectrometry/mass spectrometry （LC-MS/MS） was employed to determine the protease solution peptides,connect the peptide mass fingerprinting （PMF） via Mascot retrieval process to query protein sequence Swissprot database.Finally Western blot was employed to detect the specific serum protein markers.Results The m/z 5 816 protein marker was sieved out.And it had a high expression in renal tumor group （intensity：2128.3 ＆#177; 137.2） and a lower expression in normal children group （30.4 ＆#177; 7.8）.The difference was statistically significant （P〈0.01）.By cross test,the discriminant on the test set had a specificity of 100% and a sensitivity of 100% according to the marker combination model.A comparison of normal and trauma groups revealed the most significant difference of m/z 5 816 protein marker.And it had a high serum expression in trauma group （3674.6 ＆#177; 255.7） and a low expression in normal group （30.4 ＆#177; 7.8）.The difference was statistically significant （P〈0.01）.After screening and identification,this specific protein was thioredoxin 1 （Trx-1）.Conclusions Thioredoxin1 （Trx-1） is a protein associated with traumatic stress in pediatric nephroblastoma.
出处
《中华小儿外科杂志》
CSCD
北大核心
2014年第5期343-347,共5页
Chinese Journal of Pediatric Surgery
基金
国家自然科学基金（81172085）
