结直肠癌转移生物标记物的蛋白质组学研究

目的应用蛋白质组学技术寻找结直肠癌转移潜在标志物，研究氟尿嘧啶（5-Fu）对差异蛋白表达的影响，并初步探讨结直肠癌的转移机制。方法常规培养Lovo及SW480细胞至指数生长期后提取蛋白。采用MTF法检测5-Fu对此两种细胞的半数抑制浓度（IC50），分别用IC50的5-FU干预Lovo和SW480细胞后提取蛋白。对所提取的蛋白进一步行二维凝胶电泳，选取表达有显著差异的点进行质谱检验和生物信息学分析。采用Western blot及免疫荧光验证5-FU作用前后两种细胞显著差异蛋白的表达。结果二维凝胶电泳和质谱分析成功鉴定出11种差异蛋白．根据预设条件筛选出：核内不均匀核糖核蛋白K（hnRNP K）、蛋白质二硫键异构酶（PDI）。Western blot及免疫荧光实验显示;Lovo细胞株中hnRNPK蛋白表达较SW480高，PDI蛋白表达较SW480低：5-Fu干预后．Lovo细胞株中hnRNP K蛋白表达下调程度较SW480显著．免疫荧光强度减弱，SW480细胞株中PDI蛋白表达上调幅度较Lovo细胞显著，免疫荧光强度增强。结论hnRNP K和PDI表达在不同转移潜能结直肠癌细胞系Lovo和SW480存在显著差异，5-Fu干预后其表达有着规律性改变。

Proteomic research of biomarker of colorectal cancer metastasis

Objective To explore the potential markers of colorectal cancer metastasis and the influence of 5-FU on differentially expressed proteins by using proteomie technology, and to elucidate the mechanism of colorectal cancer metastasis. Methods Human colorectal carcinoma cell lines of different metastatic potential, Lovo and SW480 were conventionally cultured, and the protein was extracted. 50% inhibitory concentration (IC<,50) of 5-FU to these two cell lines was measured by MTT assay. Proteins of these two cell lines after intervention by 5-FU at IC<,50 were extracted, then 2-dimensional gel electrophoresis was conducted for the proteins. The differential protein spots were examined by mass spectrometry and analyzed by bioinformatics. Difference of expressed proteins in two cell lines before and after the intervention of 5-FU was validated by Western blot and immunofluorescence. Results Eleven differentially expressed proteins were identified by 2-dimensional gel electrophoresis and mass spectrometry. The hnRNP K protein and PDI were selected to be examined by Western blot and immunofluorescence. Results revealed that the expression of hnRNP K in Lovo was higher than that in SW480, while the expression of PDI was lower in Lovo. After intervention by 5-FU at IC<,50, the expression of hnRNP K in Lovo decreased more as compared to SW480, while the expression of PDI in SW480 increased more as compared to Lovo. Coulusion There are significant differences in expression of hnRNP K and PDI proteins between Lovo and SW480 cell lines, and the proteins alter regularly after 5-FU intervention.