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背景與目的:腫瘤轉移相關蛋白在腫瘤細胞轉移過程中具有重要作用,本研究在建立高轉移潛能乳腺癌細胞株LM-MCF-7的基礎上,篩選乳腺癌細胞轉移相關蛋白并探討其功能。方法:應用蛋白質組學和免疫印跡等方法比較和篩選來源相同但具有不同轉移能力的乳腺癌細胞株(MCF-7/LM-MCF-7)的蛋白質表達差異。將克隆有存活蛋白(survivin)的真核表達載體pcDNA3-Sur轉染至MCF-7細胞,通過“傷口愈合”實驗檢測存活蛋白對乳腺癌細胞遷移的影響。結果:應用蛋白質組學技術獲得8個有意義的蛋白質,分別有參與細胞骨架構成、物質代謝、細胞凋亡、蛋白折疊和蛋白質相互作用等功能。應用免疫印跡方法發現,在MCF-7細胞中nm23、p27表達水平較高,在LM-MCF-7中存活蛋白、肌球蛋白輕鏈激酶(myosinlightchainkinase,MLCK)和Bcl-2表達水平較高。“傷口愈合”實驗顯示,存活蛋白過表達可明顯增強MCF-7細胞的遷移能力。結論:不同轉移能力的乳腺癌細胞株中存在與乳腺癌轉移相關的蛋白質,存活蛋白與乳腺癌轉移有密切的關系。

建立并應用人H74 0 2肝癌細胞SCID鼠腫瘤轉移模型 ,從轉移肺組織經原代細胞培養 ,篩選并建立轉移亞細胞系M H74 0 2 ,進而運用蛋白質組學技術篩選腫瘤轉移相關蛋白 .通過二維電泳技術檢測 ,比較M H74 0 2細胞和親本H74 0 2細胞的總蛋白 ,從多個差異蛋白質點中選擇出 3個在M H74 0 2細胞中表達明顯上調的蛋白質點進行ESI QUAD TOF質譜分析 ,并在MSDB公共蛋白數據庫中進行同源比較和分析鑒定 .初步確定這 3個蛋白質分別為原肌球蛋白 (tropomyosin) ,波形纖維蛋白 (vimentin)和熱休克蛋白 70 (heatshock 70protein ,HSP70 ) .這些蛋白質參與細胞骨架構成、蛋白質折疊和蛋白質相互作用等許多正常生理活動 ,并有報道原肌球蛋白和波形纖維蛋白與腫瘤轉移有關 .利用蛋白質組學方法發現 ,在肝癌轉移亞細胞M H74 0 2中 ,原肌球蛋白、波形纖維蛋白和熱休克蛋白 70表達明顯上調 ,進一步揭示它們在腫瘤轉移中具有重要的作用 . Metastasis is the most insidious incidence of cancers.However, little is known about the molecular mechanism of tumor metastasis.A highly metastatic sub-line of H7402 hepatoma cells (termed M-H7402) was developed through screening from the lung tissue of severe combined immunodeficiency (SCID) mice injected with H7402 cells by primary cell culture, M-H7402 cell line was selected as an experimental model.Two-dimensional gel electrophoresis and mass spectrometry are combined to approach the protein profiles associated with tumor metastasis between H7402 and M-H7402 cell lines.Image analysis of Coomassie brilliant blue stained two-dimensional gels revealed that three protein spots showed significantly quantities variation.These candidate proteins were detected by ESI-QUAD-TOF mass spectrometer analysis, and searched in a publicly available MSDB database using peptide mass fingerprinting (PMF) .The results showed that they were tropomyosin, vimentin and heat shock protein 70 (HSP70) , respectively.It was reported that these proteins were involved in the normal physiological activities, such as composing cytoskeleton, protein folding and protein interactinon, etc.Recent study demonstrated that tropomyosin is an essential filament protein and vimentin expression coincides with cellular growth and cell cycle progression as well;while certain tumor cell lines express HSP-70 on cell surface.The up-regulation of tropomyosin, vimentin and heat shock protein 70 were found in M-H7402 cells, suggesting that the proteins were in