猪软骨多糖对滚瓶培养MCF-7细胞的诱导凋亡作用及其差异表达蛋白的初步研究

Apoptosis-inducing Effect of Porcine Cartilage Polysaccharide on MCF-7 Cells and Their Differentially Expressed Proteins

摘要:

本文研究了人乳腺癌细胞MCF-7在滚瓶培养形成大量细胞聚集体时，猪软骨多糖对MCF-7的诱导凋亡作用，并初步研究了其凋亡前后的差异表达蛋白。采用Hoechst 33342/PI染色于共聚焦显微镜下观察细胞的凋亡特征；Annexin V-FITC/PI检测细胞凋亡；流式细胞仪检测细胞周期的变化；并提取MCF-7细胞的全蛋白，利用二维凝胶电泳（2-DE）结合MALDI-TOF质谱技术检测差异表达蛋白。结果表明：MCF-7细胞在猪软骨多糖作用后出现典型的凋亡现象，通过流式细胞仪检测发现在猪软骨多糖作用12 h，24 h的MCF-7凋亡率分别为20.23%和44.7%；同时发现MCF-7细胞在猪软骨多糖作用后，细胞周期被阻滞在了S期；初步鉴定出2个差异表达蛋白，其中一个为肌动蛋白actin，另一个为抗氧化类蛋白PRDX6。

Abstract:

MCF-7 (human breast adenocarcinoma cell line) cells were cultured in roller bottles to investigate the apoptosis-inducing effects of porcine cartilage polysaccharide in terms of formation of large cell aggregates. Protein expression before and after apoptosis were explored. The cell apoptotic characteristics were observed by laser scanning confocal microscopy (LSCM) after Hoechst 33342/propidium iodide (PI) staining; apoptosis was detected by Annexin V-fluorescein isothiocyanate (FITC)/PI; and changes in cell cycle were analyzed by flow cytometry (FCM). Total proteins of MCF-7 were extracted and differentially expressed proteins were analyzed by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF). The results showed that the typical apoptotic characteristics of MCF-7 cells were observed after exposure to porcine cartilage polysaccharide, and FCM results showed apoptotic rates of 20.23% and 44.7% after 24- and 48-h exposure, respectively. Additionally, it was found that the cell cycle of MCF-7 cells was arrested in S phase. Moreover, two differentially expressed proteins were identified: actin and an antioxidant protein, PRDX6.