人骨肉瘤细胞株与正常成骨细胞株的比较蛋白质组学研究

Comparative proteomic analysis of proteins between human osteosarcoma and normal osteoblastic cell lines

目的探讨寻找可能作为骨肉瘤早期诊断标志物和治疗靶点的特异蛋白质。方法提取人骨肉瘤细胞株MG-63与人正常成骨细胞株hFOB1．19的总蛋白进行双向电泳，筛选两者差异蛋白质，利用基质辅助激光解吸电离飞行时间质谱分析（MALDI—TOF．MS）鉴定相关蛋白质；采用Westernblot进行体内验证，比较差异最明显的蛋白质在骨肉瘤组织和瘤旁组织的表达强度。结果二维凝胶电泳分离显示两组间差异存在2倍及以上的蛋白点有73个，其中上调的蛋白点有50个，下调的差异蛋白点有23个；选择其中差异有统计学意义的12个点行MALDI—TOF．MS质谱分析，共检测出8种蛋白；Westernblot结果显示波形蛋白在骨肉瘤组织组和癌旁组织均有表达，骨肉瘤标本中波形蛋白的表达强度显著高于对照组（P〈0．05）。结论比较蛋白质组学能很好的显示骨肉瘤细胞株与正常成骨细胞株差异表达的蛋白质；波形蛋白可能是骨肉瘤早期诊断标志物和特异性的治疗靶点。

Objective To find the special proteins to be potentiaUy used as biomarkers at early di- agnosis and treatment of osteosarcoma. Methods Total proteins obtained in MG-63 cells and osteoblastic ceils were subjected to two-dimensional electropboresis （2-DE）. Differential protein spots were character- ized by matrix assisted laser desorption/ionization-time of flight mass spectrometry （ MALDI-TOF-MS）. Fi- nally the special spot vinmentin was detected and verified by using Western blotting in osteosarcoma tissue of patients in vivo. Results The total protein profiles of two cell lines were obtained through 2-DE com- bined with softw^e ImageScanner. There were 73 protein spots detected by software Image Scanner differed in 2-fold between two cell lines, 50 spots up-regulated and 23 down-regulated. Twelve significant differenti- al spots between two cell lines were chosen to characterize by MALDI-TOF-MS. Eight proteins have been characterized. Western blotting revealed that there was expression of vinmentin in osteosarcoma tissue sam- pies, and the expression intensity of vinmentin in osteosarcoma tissue samples was significantly stronger than in controls （ P 〈 0. 05 ）. Conclusion Comparative proteomics can identify differential proteins in human osteosarcoma and normal osteoblastic cell lines, and vinmentin may be the key protein as the biomark- er at early diagnosis and treatment of osteosarcoma.