小鼠黑色素瘤转移相关蛋白的差异蛋白质组学分析*

目的:分析B16-F10黑色素移植瘤及其肺转移瘤的差异表达蛋白，以筛选黑色素瘤转移相关的分子标志。方法:应用荧光差异凝胶电泳（two-dimensional differential gel electrophoresis ，2D-DIGE）结合基质辅助激光解析电离飞行时间质谱技术（matrix assisted laser desorption ionisation time-of-flight mass spectrometry ，MALDI-TOF-MS）分离鉴定B16-F10黑色素移植瘤及其肺转移瘤的差异表达蛋白，部分差异蛋白经Real-time PCR进行mRNA 表达水平验证。结果:Decyder6.0 软件分析结果显示2D-DIGE图谱分辨率高、重复性好，30个蛋白点在实验组和对照组间存在表达差异（|Ratio| ≥2，P<0.01），经质谱分析和数据库查询鉴定出9个蛋白在实验组表达上调，包括肌红蛋白（myoglobin，MB）、波形蛋白（vimentin，VIM）、磷酸甘油激酶1（phosphoglycerate kinase 1，PGK 1）、磷酸丙糖异构酶（Triosephosphate isomerase ，TPI 或TIM）、重链结合蛋白（heavy-chain binding protein，BiP）、α- 烯醇化酶（α-enolase 或enolase 1）、β-肌动蛋白（β-actin）、γ-肌动蛋白（γ-actin）、层连蛋白结合蛋白（laminin-binding protein ），这些蛋白主要参与了细胞骨架构成、糖酵解等生物学过程。Real-time PCR结果显示糖酵解酶PGK 1 及TPI mRNA 表达水平在实验组显著高于对照组（P=0.001，0.003），变化趋势与蛋白质组学相一致。结论:小鼠黑色素瘤转移过程与多种蛋白的异常表达有关，糖酵解酶PGK 1、TPI 可能参与了黑色素瘤的转移过程。 

Differential Proteomic Analysis of Metastasis-associated Proteins in Mice Melanoma

Objective: To investigate differentially expressed protein profiles in B16-F10 grafted melanoma and its metastasis in the lung in order to identify molecular markers of melanoma metastasis. Methods: Differentially expressed proteins in B16-F10 grafted melanoma and its metastatic lesion in the lung were isolated and identified by fluorescence two-dimensional differential gel electrophoresis(2D-DIGE)coupled with matrix assisted laser desorption ionisation time-of-flight mass spectrometry(MALDI-TOF-MS).Some of identified proteins were further confirmed by Real-time PCR analysis. Results: High resolutional images of differential gel electrophoresis were obtained and 9 of 30 differentially expressed proteins (IRatiol≥2,P<0.01)were identified by MALDI-TOF-MS.The expression of Myoglobin(MB),vimentin(VIM),phosphoglycerate kinase 1(PGK1),Triosephosphate isomerase(TPI or TIM),heavy-chain binding protein(BiP),α-enolase,β-actin,γ-actin,and laminin-binding protein were up-regulated in the experimental group compared with the control group.These proteins were involved in the cytoskeletal formation,glycolysis and so on.Real-time PCR analysis showed up-regulation of mRNA expression of PGK1 and TPI in the experimental group(P=0.001 and 0.003),which was in consistent with the resuits of proteomic analysis. Conclusion: A variety of abnormally expressed proteins contribute to the metastasis of mice melanoma.Glycolytic enzymes PGK1 and TPI may be involved in this process.