摘要
背景与目的:p21是一种重要的细胞周期负性调控因子,目前对其在乳腺癌化疗过程中的作用尚不明确。本课题旨在观察将p21基因转染乳腺癌细胞后,对表柔比星敏感性的改变,以探讨新的表柔比星耐药可能的机制。方法:构建表达载体pEGFP-p21,采用脂质体转染法转入乳腺癌细胞株MCF-7中,筛选稳定表达p21的克隆;应用实时荧光定量PCR法检测转染组细胞中p21mRNA的表达,流式细胞术和Hoechst33342荧光染色法分析细胞周期分布和凋亡变化;应用MTT法检测p21基因转染前后MCF-7细胞对表柔比星敏感性的变化,实时荧光定量PCR法(real-time fluorogent quantitative PCR,RFQ-PCR)观察survivin mRNA水平的变化。结果:pEGFP-p21载体转染MCF-7细胞后,MCF-7细胞中p21mRNA的表达量明显增高,是空白对照组的155倍;转染后的细胞被阻滞于G0/G1期,但实验组与空白对照组相比,凋亡率改变的差异无统计学意义(P>0.05);表柔比星与p21基因转染联合作用时,p21可促进表柔比星对MCF-7细胞的杀伤作用,且细胞的抑制随作用时间的增加而增强,同时伴有survivin mRNA表达的降低,与表柔比星单独作用相比,差异有统计学意义(P<0.05)。结论:p21基因能增强MCF-7细胞对表柔比星的敏感性,其作用可能是通过使细胞周期发生G0/G1期阻滞及下调survivin的表达来实现的。
Background and purpose：p21 was initially identifi ed as a member of cyclin-dependent kinase inhibitor （CKI） but little was known about whether p21 level was correlated with the chemotherapy sensitivity of breast cells.In this study,we investigated the enhanced effect of p21 on the chemotherapy of breast cancer cells and explored its biological mechanism.Methods：The fusion gene of pEGFP-p21 expression vector was constructed by gene subclone.pEGFP-p21 and pEGFP-C2 control were transfected into MCF7 cells by lipofectamine and then positive clones were screened out by G418.The mRNA expression of p21 was later detected by real-time ？ uorescent quantitative polymerase chain reaction （RFQ-PCR）.Cell cycle and cell apoptosis were analyzed by ？ ow cytometry and Hoechst33342-PI staining,respectively.MCF7 cells were treated by p21 transfection and/or 0.2 μg/mL epirubicin.MTT assay was used to detect the inhibitory rate of MCF7 cell growth.The level of survivin mRNA was measured by RFQ-PCR.Results：After transfection,the mRNA level of p21 was increased by 155 folds of that in the empty control.Over expression of p21 resulted in a G0/G1 phase arrest （P0.01）,whereas there was no correlation with cell apoptosis （P0.05）.The proliferation inhibition rate was signifi cantly higher in the combination group than in the EPI treatment group,this effect was enhanced along with the increasing time of co-treatment.The mRNA level of survivin was decreased sharply in the combination group than in the EPI treatment group （P0.05）.Conclusion：p21 could possibly through the block at G0/G1 phase and down-regulation of suvivin expression enhance the effect of EPI on the proliferation inhibition of MCF7 cells.
出处
《中国癌症杂志》
CAS
CSCD
北大核心
2010年第9期646-651,共6页
China Oncology
