肾母细胞瘤血清相关炎症因子的筛选和鉴定

Discovery and identification of serum inflammation factors related to nephroblastoma

目的 探讨肾母细胞瘤血清中的相关炎症因子,与非炎症标记物共同参与肿瘤的早期诊断,同时作为肾母细胞瘤预后监测的重要血清学指标的意义.方法 收集肾母细胞瘤患儿术前血清40例（术前组）,术后2周血清35例（根治性手术29例,姑息性切除6例,术后组）,正常儿童血清50例（正常组）,炎症患儿血清50例（炎症组）.利用表面增强激光解析电离飞行时间质谱（SELDI-TOF-MS）平台在术前组与正常组、正常组与炎症组筛选特异性蛋白峰值,两组结果进行聚类分析,肿瘤组中发现炎症相关蛋白峰.经过固相萃取（SPE）和聚丙烯酰胺凝胶电泳（TRICINE-SDS-PAGE）分离、纯化目标蛋白,酶解后的肽段置入基质辅助激光解析电离飞行时间质谱（MALDI-TOF-MS）,通过Mascot搜索软件,与Swissprot数据库连接,搜索相匹配的蛋白.结果 肿瘤血清中存在m/z位于11 118.8和11 350.8两个炎症相关蛋白峰,术前组高表达（1 219.6±476.3、1 568.0±1 043.2）,正常组明显低表达（4.8±3.5、6.3±2.9）,炎症组明显高表达（2 171.4±1 247.6、3 282.0±3 656.9）.术前组与正常组、正常组与炎症组比较,差异均有统计学意义（P＜0.01）.与术前组比较,术后组2种蛋白表达均有不同程度下降（姑息性切除术后214.5±176.3和643.5±592.4,根治术后4.8±4.2和6.2±4.3）,差异有统计学意义（P＜0.01）.29例根治术后患儿表达强度分别为4.8±4.2和6.2±4.3,与正常组比较,差异无统计学意义（P＞0.05）.6例姑息性切除术后表达强度214.5±176.3、643.5±592.4,与正常组比较,差异有统计学意义（P＜0.01）.m/z 11 118.8和11 350.8的蛋白峰经鉴定为巨噬细胞移动抑制因子（MIF）和趋化因子（CXCL7）.结论 肾母细胞瘤血清确实存在相关炎症因子,预后越好,表达量越低.与前期实验结果非炎症标记物类载脂蛋白C-I共同参与疾病的早期诊断,对后期研究炎症因子与肿瘤关系具有指导意义.

Objective To explore the serum inflammation factors in nephroblastoma,examine the feasibility of an early diagnosis with non-inflammatory biomarkers and seek important serum markers for recovery monitoring of nephroblastoma.Methods The serum samples of 40 preoperative nephroblastoma cases,35 postoperative cases（2 weeks postoperation） including 29 radical operation cases and 6 cases of palliative excision,50 healthy and 50 inflammatory cases were collected.SELDI-TOF-MS was employed for screening inflammatory biomarkers in preoperative group.Targeted proteins were purified with SPE and TRICINE SDS-PAGE.After In-Gel digestion,peptide fragments were loaded into MALDI-TOF-MS.Mascot software was searched and SwissProt database connected.After comparing and matching,target proteins were obtained.Results Two inflammatory protein peaks of m/z 11 118.8 and 11 350.8 were obtained through SELDI-TOF-MS in preoperative group.There was a high expression in preoperative group（1 219.6 ± 476.3 and 1 568.0 ± 1 043.2）.There were a low expression in normal control group （4.8 ± 3.5 and 6.3 ± 2.9） and a high expressien in inflammation group （2 171.4 ± 1 247.6 and 3 282.0 ± 3 656.9）.The results between preoperative and normal control,inflammation and normal control groups had statistical differences（P＜0.01）.In 29 radical operation cases,the expression intensity was 4.8 ± 4.2 and 6.2 ± 4.3.The result had no statistical difference comparing with normal control group（P＞0.05）.In 6 cases of palliative excision,the expression intensities were 214.5 ± 176.3 and 643.5 ± 592.4 and they were lower than those of preoperative group（P＜0.01）.The result had statistical significance as compared to normal control group（P＜0.01）.And m/z 11 118.8 and 11 350.8 protein peaks were identified as macrophage migration-inhibitory factor （MIF） and CXCL7.Conclusions Serum inflammation factors exist in nephroblastoma.Along with previously identified non-inflammation biomarker APOC-I,they may aid an early diagnosis of disease.