人Mad2基因正义真核表达质粒的构建及其对人卵巢癌细胞A2780紫杉醇敏感性的影响

Construction of human Mad2 sense eukaryotic expression vector and its effect on the sensitivity to paclitaxel in human ovarian cancer cell line A2780

背景与目的:紫杉醇是一种高效广谱的抗肿瘤药物,已常规应用于卵巢癌的化疗。但近年来随着耐药的发生,紫杉醇耐药已成为影响肿瘤化疗预后的主要难题,有研究显示紫杉醇药效的发挥与纺锤体检测点功能完整性相关。本研究通过构建人纺锤体检测点蛋白Mad2基因正义真核表达质粒,旨在探讨Mad2蛋白对人卵巢癌A2780细胞株紫杉醇敏感性的影响。方法:构建pEGFP-Mad2正义表达质粒,通过脂质体LipofectamineTM 2000将其转入体外培养的人卵巢癌细胞株A2780中,应用RT-PCR、Western blot等方法分析相关基因和蛋白水平的变化情况;通过流式细胞术比较稳筛细胞对紫杉醇敏感性的改变情况。结果:成功构建了pEGFP-Mad2正义表达质粒;并筛选出稳定高表达Mad2的细胞株。RT-PCR、Western blot检测均显示稳定筛出的pEGFP-Mad2/A2780细胞株中Mad2呈高表达。流式细胞术检测结果显示,紫杉醇作用24或48 h后,对照组pEGFP-N1/A2780的细胞凋亡率分别为12.34%和58.76%,而pEGFP-Mad2/A2780组细胞凋亡率则分别为27.32%和87.53%,差异有统计学意义(P<0.05)。结论:pEGFP-Mad2正义表达质粒能够有效上调A2780细胞中Mad2基因mRNA和蛋白的表达,并增加人卵巢癌细胞A2780对紫杉醇化疗的敏感性。

Background and purpose：Paclitaxel,one of the broadest spectrum anticancer agents,is effective in the chemotherapy of patients with ovarian cancers which kills cancer cells by disrupting microtubule dynamics.With the increasing of chemotherapy resistance in patients,paclitaxel resistance is the major factor involved in poor response and reduced overall survival.Base on studies in recent years,chemoresistance to paclitaxel treatment was mentioned in relation to the function of spindle assembly checkpoint（SAC）.In this study,we constructed the human SAC protein Mad2 sense eudaryotic expression plasmid and investigated the relationship between the high-expression level of Mad2 and the paclitaxel sensitivity in the human ovarian cancer cell line A2780.Methods：pEGFP-Mad2 plasmid was constructed and then transfected into A2780 cell line by the Lipofectamine 2000,the empty vector pEGFP-N1 was the control RT-PCR and Western blot were used to analyse the Mad2 gene mRNA and protein levels.The difference of the cell sensitivity to paclitaxel between pEGFP-N1/A2780 and pEGFP-Mad2/A2780 stable cell lines was tested with FACS.Results：The human Mad2 sense eukaryotic expression plasmid was constructed successfully.RT-PCR and Western blot analyses revealed that pEGFP-Mad2/A2780 stable cell line expressed higher levels of Mad2 mRNA and protein compared to controlp EGFP-N1/A2780 cells.Flow cytometry showed that the rates of apoptosis of control pEGFP-N1/A2780 cells were 12.34% and 58.76% after the paclitaxel treatment of 12 or 24 h.However,in pEGFP-Mad2/A2780 stable cells,the rates of apoptosis were 27.32% and 87.53% with 12 or 24 h paclitaxel treatment.The sensitivity of pEGFP-Mad2/A2780 cells to paclitaxel was increased compared with the control ones（P0.05）.Conclusion：Transfection of the pEGFP-Mad2 plasmid could increase the expression level of Mad2,which could make the A2780 cells more sensitive to paclitaxel.