CAJ | 학술논문

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Bubl是纺锤体检查点的重要组成部分.本研究旨在探讨Bubl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Bubl基因短发卡状RNA真核表达载体pEGFP-Bubl-shRNA,以脂质体Lipofectamine~(TM)2000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-Cl/A2780及A2780组相比,pEGFP-Bubl-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-Bubl-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1 μ mol/L处理细胞24及48 h后,与pEGFP-Cl/A2780及A2780组相比,该组细胞凋亡率明显降低(P＜0.05),G_2/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-Bubl-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:Bubl基因的下调可导致人卵巢癌A2780细胞对紫杉醇敏感性及G_2/M期细胞比例下降.pEGFP-Cl-shBubl质粒的成功构建,为研究Bubl基因的功能及定位的研究提供了进一步的实验条件.
배경여목적:기왕연구제시,자삼순류약물작용적발휘유뢰우공능완정적방추체검사점,이Bubl시방추체검사점적중요조성부분.본연구지재탐토Bubl단발잡상RNA진핵표체재체은정전염대인란소암A2780세포자삼순민감성급세포주기적영향.방법:구건Bubl기인단발잡상RNA진핵표체재체pEGFP-Bubl-shRNA,이지질체Lipofectamine~(TM)2000포과공질립전염체외배양적인란소암세포주A2780후진행은정사선.응용RT-PCR、Western인적법분석목적기인mRNA급기단백수평적표체정황:MTT법、류식세포술등방법검측전염전후,A2780세포대자삼순민감성급기세포주기적변화;Hoechst33342염색법검측세포분렬지수.결과:여대조조pEGFP-Cl/A2780급A2780조상비,pEGFP-Bubl-ShRNA/A2780조세포중mRNA화단백수평적표체균명현하강,기차이구유통계학의의(P＜0.05);MTT검측결과제시,전염pEGFP-Bubl-shRNA질립후세포대자삼순적민감성명현강저,여대조조상비,차이구유통계학의의(P＜0.05);류식세포술검측결과현시,자삼순1 μ mol/L처리세포24급48 h후,여pEGFP-Cl/A2780급A2780조상비,해조세포조망솔명현강저(P＜0.05),G_2/M기세포비례하강,차이구유통계학의의(P＜0.05);Hoechst33342염색제시,여대조조상비,pEGFP-Bubl-shRNA/A2780조적분렬지수(MI)명현강저,차이구유통계학의의(P＜0.05).결론:Bubl기인적하조가도치인란소암A2780세포대자삼순민감성급G_2/M기세포비례하강.pEGFP-Cl-shBubl질립적성공구건,위연구Bubl기인적공능급정위적연구제공료진일보적실험조건.
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-Bubl-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 2000~(TM).The nontransfected 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 bv 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-Cl/A2780 group(P＜0.05).The sensitivity of the pEGFP-Bubl-shRNA/A2780 group was significantly lower than the non-transfccted and pEGFP-Cl/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 G_2/M cells in human ovarian cancer cell line A2780.