Perego Roberto A, Bianchi Cristina, Corizzato Matteo, Eroini Barbara, Torsello Barbara, Valsecchi Cristina, Di Fonzo Andrea, Cordani Nicoletta, Favini Paolo, Ferrero Stefano, Pitto Marina, Sarto Cecilia, Magni Fulvio, Rocco Francesco, Mocarelli Paolo
Department of Experimental, Environmental Medicine and Medical Biotechnologies, Milano-Bicocca University, Monza, Italy.
J Proteome Res. 2005 Sep-Oct;4(5):1503-10. doi: 10.1021/pr050002o.
Renal cell carcinoma (RCC) tissue is composed of a mixture of neoplastic and normal cells, which complicate proteome analysis. The aim of our study was to investigate whether it is feasible to establish primary cell cultures of RCC and of renal cortex maintaining the tissue phenotype along with a more homogeneous and enriched cytological material. Fourteen (82.3%) primary cultures from 17 surgical cases were established and characterized by morphology, growth rate, immunocytochemistry, and molecular analysis performed by Real-time PCR, Western blotting, two-dimensional electrophoresis (2-DE), and mass spectrometry. Cultures showed >90% cytokeratine-positive epithelial cells. In primary tumor cultures, the molecular phenotype of manganese superoxide dismutase and heat shock protein 27 was the same as that found in tumor tissues with overexpression and increased number of isoforms. Moreover, 27 out 28 specific proteins and their isoforms, present in spots excised from 2-DE gel of cortex or RCC cultures, corresponded to those identified on the 2-DE tissue cortex reference map, suggesting that these primary cultures retain the proteomic profile of the corresponding tissues.
肾细胞癌(RCC)组织由肿瘤细胞和正常细胞混合组成,这使得蛋白质组分析变得复杂。我们研究的目的是探讨建立RCC和肾皮质原代细胞培养物,并维持组织表型以及获得更均匀和丰富的细胞学材料是否可行。从17例手术病例中建立了14个(82.3%)原代培养物,并通过形态学、生长速率、免疫细胞化学以及通过实时PCR、蛋白质印迹、二维电泳(2-DE)和质谱进行的分子分析对其进行了表征。培养物显示>90%的细胞角蛋白阳性上皮细胞。在原发性肿瘤培养物中,锰超氧化物歧化酶和热休克蛋白27的分子表型与在肿瘤组织中发现的相同,具有过表达和同工型数量增加的情况。此外,从皮质或RCC培养物的2-DE凝胶上切下的斑点中存在的28种特定蛋白质及其同工型中的27种,与在2-DE组织皮质参考图谱上鉴定出的蛋白质相对应,这表明这些原代培养物保留了相应组织的蛋白质组图谱特征。