高恶性膀胱移行细胞癌的蛋白质组学研究及差异蛋白mortalin的表达验证

摘要

Objective: To investigate the differences in protein expression profiles between highly malignant bladder transitional cell carcinoma (BTCC) and normal urothelial cells and to verify the differentially expressed proteins that might be potential and valuable biomarkers for BTCC.Methods: The proteins expressed in highly malignant BTCC and normal urothelial cells were separated by hand microdissection combining two-dimensional electrophoresis (2-DE), and then identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS).Semi-quantitative RT-PCR and immunohistochemistry were employed for further verification, Results: A total of 11 differentially expressed proteins were identified and five of them including cytoskeletal 7, 60 kDa heat shock protein, cytoskeletal 8, annexin A5 and prohibitin were reported possibly correlated with the development of BTCC.The other 6 proteins were mortalin, 14-3-3 protein epsilon, protein disulfide-isomera-se A6, annexin A4, desmin and tubulin beta-3 chain.Semi-quantitative RT-PCR and immunohistochemistry showed that the screened protein mortalin was up-regulated in highly malignant BTCC ( P ＜ 0.01 ).Conclusion: The protein profile of highly malignant BTCC is obviously different from that of normal urothelial cells, indicating involvement of various distinct proteins in bladder carcinogenesis.The identified protein mortalin might be a potential and valuable biomarker for bladder transitional cell carcinoma.%目的:寻找高恶性膀胱移行细胞癌(BTCC)和正常膀胱黏膜的差异表达蛋白,并对筛选出的有潜在应用价值的差异蛋白进行再验证,试图发现与高恶性膀胱移行细胞癌发生、进展相关的生物学标记物.方法:联合手工显微切割、双向电泳(2-DE)及基质辅助激光解吸/电离串联飞行时间质谱(MALDI-TOF/TOF-MS)对高恶性膀胱移行细胞癌与癌旁相应正常膀胱移行上皮进行蛋白质的分离和鉴定.并采用半定量RT-PCR技术和免疫组织化学技术对鉴定出的差异蛋白进行再验证.结果:共鉴定出11个差异表达蛋白,其中细胞角蛋白7(CK-7)、细胞角蛋白8(CK-8)、热休克蛋白60(HSP60)、膜联蛋白Ⅴ、抑制素这5个蛋白曾在文献中报道过,其表达失调与BTCC的发生发展可能相关;其余6个蛋白,包括mortalin、14-3-3ε蛋白、蛋白质二硫键异构酶A6前体蛋白、膜联蛋白Ⅳ、结蛋白、微管蛋白β4,目前尚未见到与BTCC的相关报道.半定量RT-PCR和免疫组织化学染色结果显示mortalin在高恶性膀胱移行细胞癌中mRNA和蛋白表达水平均高于正常膀胱移行上皮(P<0.01).结论:高恶性BTCC的蛋白表达谱与相应正常的膀胱移行上皮相比有明显差异,由此表明膀胱移行细胞癌的发生是一个多基因多途径参与的复杂过程.mortalin蛋白在高恶性膀胱移行细胞癌中的表达明显上调与双向电泳的结果一致,mortalin可能成为一个潜在的、有应用价值的膀胱移行细胞癌的标记物.