CAJ | 학술논문

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MTT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hnRNP K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.
목적 응용단백질조학기술심조결직장암전이잠재표지물,연구불뇨밀정(5-FU)대차이단백표체적영향,병초보탐토결직장암적전이궤제.방법 상규배양Lovo급SW480세포지지수생장기후제취단백.채용MTT법검측5-FU대차량충세포적반수억제농도(IC50),분별용IC50적5-FU간예Lovo화SW480세포후제취단백.대소제취적단백진일보행이유응효전영,선취표체유현저차이적점진행질보검험화생물신식학분석.채용Western blot급면역형광험증5-FU작용전후량충세포현저차이단백적표체.결과 이유응효전영화질보분석성공감정출11충차이단백,근거예설조건사선출:핵내불균균핵당핵단백K(hnRNP K)、단백질이류건이구매(PDI).Western blot급면역형광실험현시:Lovo세포주중hnRNP K단백표체교SW480고,PDI단백표체교SW480저;5-Fu간예후,Lovo세포주중hnRNP K단백표체하조정도교SW480현저,면역형광강도감약,SW480세포주중PDI단백표체상조폭도교Lovo세포현저.면역형광강도증강.결론 hnRNP K화PDI표체재불동전이잠능결직장암세포계Lovo화SW480존재현저차이,5-FU간예후기표체유착규률성개변.
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.