肾母细胞瘤瘤体中差异性炎症因子的鉴定及其临床意义

Identification of differential inflammation factors in nephroblastoma tissue and clinical significance

目的 利用蛋白组学技术研究肾母细胞瘤瘤体存在的差异性炎症因子,分析其与肿瘤的临床分期、病理分型、淋巴结转移及血管侵犯的相关性.方法 收集2010年1月至2014年12月收治的肾母细胞瘤患儿的瘤体（瘤体组）40例,肿瘤临床分期Ⅰ期6例,Ⅱ期12例,Ⅲ期13例,Ⅳ期9例;预后良好型37例,预后不良型3例;淋巴结转移17例,无淋巴结转移23例;血管侵犯9例,无血管侵犯31例.同时收集肿瘤近端（距肿瘤边缘1 cm）肾组织35例、肿瘤远端（距肿瘤边缘5 cm）肾组织25例.通过表面增强激光解吸电离飞行时间质谱技术筛选3组间的差异性蛋白峰,对瘤体中存在的高表达蛋白峰通过固相萃取技术和聚丙烯酰胺凝胶电泳进行分离、纯化,胶内酶解后的肽段混合物置入色谱串联质谱,根据氨基酸序列鉴定目标蛋白身份.根据瘤体中炎症蛋白峰的表达强度,在临床分期、病理分型、淋巴结转移及血管侵犯各组内进行比较分析.结果 肿瘤组织高表达的蛋白峰中,m/z12138和m/z13462分别被鉴定为巨噬细胞移动抑制因子和中性粒细胞激活蛋白,两者在瘤体组的表达量（1437.8±997.3和1730.4±1147.8）高于肿瘤近端肾组织（952.6±591.2和1031.1±1120.8）及肿瘤远端肾组织（315.4±296.5和114.7±118.9）,3组比较差异有统计学意义（P＜0.001）.瘤体组中m/z12138和m/z13462的表达量按不同病理特征分组进行比较：临床分期Ⅰ期（678.8±189.0和746.2±238.7）,Ⅱ期（664.0±202.0和1180.7±404.9）,Ⅲ期（1524.7±407.9和2160.4±1252.3）,Ⅳ期（2850.2±861.2和2498.4±1290.5）;预后良好型（1271.7±809.2和1553.3±991.4）,预后不良型（3487.2±166.2和3915.1±507.3）;淋巴结转移组（2207.1±961.7和2569.5±1285.2）,无淋巴结转移组（869.2±474.6和1110.2±433.6）;血管侵犯组（2850.2±861.2和2498.4±1290.5）,无血管侵犯组（1027.8±521.3和1507.5±1019.9）,各病理特征分组内比较差异均有统计学意义（P＜0.001）.结论 炎症因子巨噬细胞移动抑制因子和中性粒细胞激活蛋白高表达于肾母细胞瘤瘤体中,与肿瘤的临床分期、病理分型、淋巴结转移、血管侵犯具有相关性.

Objective To identify the differential inflammation factors in nephroblastoma tissue using proteomics technology and analyze its relationship with clinical stage,pathological phenotype,lymph node metastasis,vascular invasion.Methods From Jan 2010 to Dec 2014,nephroblastoma tumor tissues from 40 patients were obtained.Meanwhile,the 35 tissue near proximal kidney and 25 tissues distal kidney were also obtained.The classification of clinical stage included Ⅰ stage in 6 cases,Ⅱ stage in 12 cases,Ⅲ stage in 13 cases and Ⅳ stage in 9 cases.Other characters contained good prognosis type in 37 case,poor prognosis type in 3 cases,lymphatic metastasis in 17 cases,no sign of lymphatic metastasis in 23 cases,vascular invasion in 9 cases and non-vascular invasion in 31 cases.The SELDI-TOF-MS was used for screening differential protein peaks among three groups.Then,SPE and TRICINE-SDS-PAGE were used to separate and purificate the protein,which showed high peaks expression in tumor tissue,respectively.After in-gel digestion,we received the identification of targeted proteins according to sequence information through Nano-LC-MS/MS.Finally we compared differential expression of inflammatory peaks in different groups of clinical stage,pathological type,lymph node metastasis and vascular invasion.Results All the peaks high expression in tumor tissue,m/z12138 and m/z 13462 are identified as MIF and NAP-2.Expression of two protein peaks in tumor tissue（1437.8 ＋ 997.3,1730.4 ＋ 1147.8） is higher than those in proximal tissue （952.6 ＋ 591.2,1031.1 ＋ 1120.8） and in distal tissue（315.4 ＋ 296.5,114.7 ＋ 118.9）,which showed the significant difference （P 〈 0.001）.According to the clinic stage classification,the expression of those protein were 678.8 ＋ 189.0,746.2 ＋ 238.7 in stage Ⅰ,664.0 ＋ 202.0,1180.7 ＋ 404.9 in stage Ⅱ,1524.7＋407.9,2160.4 ＋ 1252.3 in stage Ⅲ and 2850.2 ＋ 861.2,2498.4 ＋ 1290.5 in stage Ⅳ.Based on the other characters,expression of those protein were the 1271.7 ＋ 809.2,1553.3 ＋ 991.4 in good prognosis type,3487.2 ＋ 166.2,3915.1 ＋507.3 in poor prognosis type,2207.1 ＋961.7,2569.5 ＋ 1285.2 in lymph node metastasis,869.2 ＋ 474.6,1110.2 ＋ 433.6 in non-lymph node metastasis,2850.2 ＋ 861.2,2498.4 ＋1290.5 in vascular invasion and 1027.8 ＋ 521.3,1507.5 ＋ 1019.9 in non-vascular invasion.All the comparison results have significant statistical difference （P 〈 0.001）.Conclusion MIF and NAP-2significantly increase in nephroblastoma tumor tissue.Meanwhile,there was obvious relationship between those protein with clinical stage,pathological type,lymph node metastasis and vascular invasion.