人骨肉瘤细胞和成骨细胞的蛋白质组学分析

Comparative proteomic analysis of human osteosarcoma cell and primary cultured osteoblastic cell

背景与目的:骨肉瘤是青少年常见的恶性肿瘤,由于大多数患者发现时疾病已属晚期,虽然手术可以改善患者局部症状,但5年生存率低,所以治疗骨肉瘤的关键是寻找肿瘤特异性靶点,研制出针对性强的化疗药物。近年来,蛋白质组学的迅速发展,为发现肿瘤标志物提供了一种新方法。本研究采用比较蛋白质组学理论和技术,筛选在骨肉瘤细胞中差异表达的蛋白,以期发现可能用于早期诊断和治疗骨肉瘤的标志蛋白。方法:将人骨肉瘤细胞株SaOS-2细胞和成骨细胞培养后,经裂解提取蛋白,进行双向凝胶电泳,电泳图像扫描后应用ImageMaster 2D图像分析软件进行比较分析,找到在骨肉瘤细胞中差异表达明显的蛋白质点,用MALDI-TOF-MS对这些差异表达的蛋白质点进行鉴定。为了验证蛋白质组学实验的结果,我们取8例临床确诊为骨肉瘤患者的手术切除标本,对鉴定出的3个蛋白质进行Western blot实验。结果:双向凝胶电泳图像分析发现有13个蛋白质点在两组细胞中有显著差异(P<0.05)。质谱分析并经过数据库检索后鉴定出在骨肉瘤细胞中热体克蛋白70(heat shock protein 70,Hsp70)、肌动成帽蛋白(actin capping protein)、ATP合成酶、线粒体热休克蛋白75(mitochondrial heat shock protein 75,Mthsp75)、泛醇细胞色素-c还原酶(ubiquinol-cytochrome-c reductase complex core proteinⅠ,UQCRC1)、Ras相关核蛋白、UCH-L1和PRDX4蛋白明显升高;丙酮酸脱氢酶(pyruvate dehydrogenase E1,PDH E1)、KIAA0088、抑制素(prohibitin,PHB)和锚定蛋白(Annexin V)表达明显降低。Western blot实验证实UQCRC1、UCH-L1和PRDX4蛋白在骨肉瘤细胞中明显升高。结论:蛋白质组学分析能很好地显示骨肉瘤细胞与成骨细胞之间差异表达的蛋白,这些鉴定出的蛋白质可以为进一步研究骨肉瘤的病理生理学提供新的分子标志物,并为研制新的治疗药物提供依据。

Background and purpose:At present,osteosarcoma is one of the most common malignant bone tumors in adolescents.Most patients are diagnosed at the advanced stage,making its 5-year survival rate very low.Therefore,finding the special target protein may improve prognosis for patients with osteosarcoma.In recent years,with the development of proteomics,finding tumor markers has become easier.This study applied the comparative proteomic theory and its techniques in order to find differently expressed proteins between the osteosarcoma cells and human primary cultured osteoblastic cells.These proteins can be biomarkers for early diagnosis and treatment of osteosarcoma.Methods:Both osteosarcoma cells SaOS-2 and human primary cultured osteoblastic cells were collected and split to extract the protein.Then 2-DE electrophoresis was conducted.The 2-DE maps were visualized after silver staining and analyzed by the ImageMaster 2D software.The differently expressed proteins were then identified using MALDI-TOF-mass spectrometry.Western blot was used to verify the results from the proteomic analyses of 8 patients with osteosarcoma.Results:Thirteen protein spots were significantly different between osteosarcoma and osteoblastic cells in the 2-DE maps.After mass spectroscopic identification and data base searches,we found that in osteosarcoma cells,the level of heat shock protein 70(HSP70),actin capping protein,ATP synthase,mitochondrial heat shock protein 75(Mthsp75),ubiquinol-cytochrome-c reductase complex core protein Ⅰ(UQCRC1),Ras-related nuclear protein,UCH-L1 and PRDX4 were all elevated.However,the level of pyruvate dehydrogenase E1(PDH E1),KIAA0088,prohibitin(PHB),and Annexin V all decreased.Subsequent Western blot analyses of UQCRC1,UCH-L1 and PRDX4 in osteosarcoma tissues confirmed the results obtained by the proteomic analyses.Conclusion:Proteomic analysis can identify protein variances in osteosarcoma as well as provide probable new biomarkers that are correlated with the biological behavior of osteosarcoma.