短发卡状RNA抑制人Bub1基因表达及其对人卵巢癌A2780细胞药物敏感性和周期的影响  

Effects of pEGFP-Bub1-shRNA plasmid on cell cycle and paclitaxel-sensitivity in human ovarian cancer ceIl line A2780

作　　者：周婷[1] 翁丹卉[1] 王世宣[1] 卢运萍[1] 马丁[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院妇产科,湖北武汉430030

出　　处：《中国癌症杂志》2010年第3期161-166,共6页China Oncology

基　　金：国家海外青年学者合作研究基金项目(项目编号:30528012);国家973重点基础研究发展计划资助项目(项目编号:2009CB521800)

摘　　要：背景与目的:既往研究提示,紫杉醇类药物作用的发挥有赖于功能完整的纺锤体检查点,而Bub1是纺锤体检查点的重要组成部分。本研究旨在探讨Bub1短发卡状RNA真核表达载体稳定转染对人卵巢癌A2780细胞紫杉醇敏感性及细胞周期的影响。方法:构建Bub1基因短发卡状RNA真核表达载体pEGFP-Bub1-shRNA,以脂质体LipofectamineTM2000包裹空质粒转染体外培养的人卵巢癌细胞株A2780后进行稳定筛选。应用RT-PCR、Western印迹法分析目的基因mRNA及其蛋白水平的表达情况;MTT法、流式细胞术等方法检测转染前后,A2780细胞对紫杉醇敏感性及其细胞周期的变化;Hoechst33342染色法检测细胞分裂指数。结果:与对照组pEGFP-C1/A2780及A2780组相比,pEGFP-Bub1-ShRNA/A2780组细胞中mRNA和蛋白水平的表达均明显下降,其差异具有统计学意义(P<0.05);MTT检测结果提示,转染pEGFP-Bub1-shRNA质粒后细胞对紫杉醇的敏感性明显降低,与对照组相比,差异具有统计学意义(P<0.05);流式细胞术检测结果显示,紫杉醇1μmol/L处理细胞24及48h后,与pEGFP-C1/A2780及A2780组相比,该组细胞凋亡率明显降低(P<0.05),G2/M期细胞比例下降,差异具有统计学意义(P<0.05);Hoechst33342染色提示,与对照组相比,pEGFP-Bub1-shRNA/A2780组的分裂指数(MI)明显降低,差异具有统计学意义(P<0.05)。结论:Bub1基因的下调可导致人卵巢癌A2780细胞对紫杉醇敏感性及G2/M期细胞比例下降。pEGFP-C1-shBub1质粒的成功构建,为研究Bub1基因的功能及定位的研究提供了进一步的实验条件。Background and purpose： Previous studies have shown that Bubl was a critical component of the spindle checkpoint. Paclitaxel sensitivity was considered to be dependent on the functionality of this spindle checkpoint. This study investigated the effects of pEGFP-Bub 1-shRNA plasmid stably transfected into the cell cycle and its sensitivity in human ovarian cancer cell line A2780. Methods： After the pEGFP-Bubl-shRNA plasmid and empty plasmids were constructed, they were transfected into A2780 cells by the Lipofectamine 2000TM. The non- transfected cells were the control. RT-PCR and Western blotting were used to determine the target gene and protein expression. The rate of proliferation inhibition was tested by an MTT assay, apoptosis and cell cycles were determined by flow cytometry, and the mitotic index was determined by Hoechst33342 dye. Results： RT-PCR and Western blotting showed that pEGFP-Bubl-shRNA/A2780 group displayed a low expression of Bubl compared to the A2780 and pEGFP-C1/A2780 group （P〈0.05）. The sensitivity of the pEGFP-Bubl-shRNA/A2780 group was significantly lower than the non-transfected and pEGFP-C1/A2780 cells （P〈0.05）. Flow cytometry revealed that the rates of G2/M phase and apoptosis were significantly lower in the pEGFP-Bubl-shRNA/A2780 group than those in the control group （P〈0.05）. Conclusion： Bubl plays an important role in the paclitaxel treatment. A down-regulation of Bubl could reduce the drug sensitivity and rate of G2/M cells in human ovarian cancer cell line A2780.

关 键 词：卵巢癌 Bub1基因 紫杉醇 耐药 

分 类 号：R73-361[医药卫生—肿瘤]