应用蛋白质组学技术研究反义Cyclin B1的抗肿瘤作用机制  

Mechanism Study of Antisense Cyclin B1 in Tumorigenesis Inhibition Using Proteomic Technique

作　　者：杨涛[1] 张伶[1] 严飞[1] 仝爱平[1] 刘新宇[1] 刘涣义[1] 黄灿华[1] 

机构地区：[1]四川大学华西医院生物治疗国家重点实验室,四川成都610041

出　　处：《癌症》2007年第3期264-269,共6页Chinese Journal of Cancer

摘　　要：背景与目的:先前研究发现采用反义技术下调cyclin B1基因表达水平,可产生抗肿瘤的作用,但其抗肿瘤机制有待进一步研究。本研究旨在利用比较蛋白质组学的方法来探讨反义cyclin B1抗肿瘤作用的分子机制。方法:首先用含有小鼠cyclin B1反义全长cDNA的重组质粒转染小鼠CT26结肠癌细胞,经双向凝胶电泳(2-DE)分离转染组和未转染组的总蛋白,再用PDQuest图像分析识别2-DE胶图像,然后对差异表达的蛋白质点进行基质辅助激光解吸电离飞行时间(MALDI-TOF-MS)质谱分析,再经Mascot软件搜索数据库鉴定这些差异蛋白。最后根据被鉴定肽段的可信度,从这些蛋白质中选出肽段可信度最高的2个,用Western blot对其丰度差异进行了验证。结果:初步鉴定出7个差异表达蛋白,其中Axin2、CCTθ、DR5和HPCM27蛋白质在实验组上调,RFP17、mKIAA1195及LOC77035蛋白在实验组下调,而可信度最高的Axin2和DR5的蛋白质表达丰度差异为Western blot所证实。结论:多种蛋白质在反义cyclin B1后表达有明显差异,这些差异蛋白质参与细胞的增殖、分化、迁移、凋亡及转录调控等多条信号通路,提示反义cyclin B1的抗肿瘤机制可能与上述蛋白质的作用有关。BACKGROUND ＆ OBJECTIVE： Previous researches showed that down-regulating the expression of cyclin B1 in tumor cells by RNA interference may inhibit tumorigenesis, but the mechanism remains to be clarified. This study was to reveal the molecular mechanism of antisense cyclin B1 in tumorigenesis inhibition by comparative proteomic technique. METHODS： A recombinant plasmid containing the full-length antisense cDNA of mouse cyclin B1 was transfected into mouse colon carcinoma cell line CT26. Total proteins of transfected cells and control cells were extracted and separated by two-dimensional gel electrophoresis （2-DE）, The differential expression proteins were analyzed with PDQuest software, and identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS） and Mascot database searching. The 2 differential proteins with the highest confidence of the peptides were selected and verified by Western blot. RESULTS： Seven differentially expressed proteins were identified. Axin2, CCTθ, DR5, and HPCM27 were up-regulated in transfected cells, while RFP17, mKIAA1195, and LOC77035 were down-regulated. The expression abundance differences of Axin2 and DR5, with the highest confidence, were verified by Western blot. CONCLUSIONS. Several proteins expressed differentially in CT26 cells after transfection of antisense cyclin B1, which take part in some signal pathways in cell proliferation, differentiation, migration, apoptosis, and transcriptional control. The antitumor effect of antisense cyclin B1 may relate to the interplay of the above proteins.

关 键 词：反义cyclin B1 抑瘤机制 双向电泳 基质辅助 激光解吸电离飞行时间质谱 

分 类 号：R73-3[医药卫生—肿瘤]