芳香化酶抑制剂来曲唑诱导乳腺癌细胞凋亡实时观测模型的建立

摘要

目的:构建芳香化酶过表达的乳腺癌细胞模型以及芳香化酶抑制剂来曲唑诱导乳腺癌细胞凋亡的实时观测模型.方法:采用慢病毒包被介导基因转导方法,构建凋亡荧光指示蛋白VC3AI稳定表达的MCF-7-VC3AI和ZR7530-VC3AI工具细胞系,同时将该细胞系过表达芳香化酶,进而构建来曲唑诱导乳腺癌细胞凋亡实时观测模型.采用实时荧光定量PCR、蛋白质印迹法检测和验证细胞中芳香化酶表达,通过MTT法检测细胞增殖.分别观察过表达芳香化酶的MCF-7-VC3AI、ZR7530-VC3AI细胞在雄激素和雌激素作用下的体外增殖能力,以及来曲唑作用下细胞的增殖能力.结果:实时荧光定量RCR检测结果显示,过表达芳香化酶MCF-7-VC3AI、ZR7530-VC3AI细胞模型中芳香化酶mRNA的表达水平明显高于MCF-7-VC3AI、ZT7530-VC3AI细胞.蛋白质印迹法结果显示,两种细胞模型中,芳香化酶蛋白的表达水平明显升高.MTT法检测结果显示,过表达芳香化酶的细胞模型在雄激素和雌激素刺激下增殖加快.100 nmol/L雄激素作用下,过表达芳香化酶MCF-7-VC3AI细胞的增殖率约为对照组的1.2倍(P<0.01),而ZR7530-VC3AI细胞的增殖率约为对照组的1.5倍(P<0.01).来曲唑以浓度依赖方式抑制雄激素诱导的细胞增殖.10 μmol/L来曲唑作用下,过表达芳香化酶MCF7-VC3AI细胞增殖率为对照组的80%,而过表达芳香化酶ZR7530-VC3AI细胞增殖率为对照组的68%.结论:成功建立了来曲唑诱导乳腺癌细胞凋亡的实时观测模型,为研究来曲唑的作用机制奠定了重要的实验基础.%Objective: To construct an aromatase-overexpressing breast cancer cell model and observe the real-time apoptosis of breast cancer cells induced by the aromatase inhibitor, letrozole. Methods: The lentivirus-mediated gene transfection method was used to construct the MCF-7-VC3AI and ZR7530-VC3AI cell lines,which stably expressed the apoptotic fluorescent indicator protein VC3AI.Simultaneously,letrozole-induced apoptosis models of the MCF-7-VC3AI and ZR7530-VC3AI breast cancer cell lines were also constructed.Real-time quantitative PCR(qPCR)and Western blot methods were used to detect the mRNA and protein levels of aroma-tase in the cells.Cell proliferation ability was measured using MTT.The proliferation of cells in vitro under testosterone,estradiol,or letrozole combined with testosterone treatments were also observed.Results:qPCR results showed that the expression of the aroma-tase mRNA was significantly higher in both the MCF-7 and ZR7530 cell models when compared to the MCF-7-VC3AI and ZR7530-VC3AI cell models.Western blot results showed that the expression of the aromatase protein was significantly increased in both cell models. MTT assay results showed that the proliferation of a cell model could be promoted by testosterone and estrogen stimulation.Under 100 nmol/L testosterone,the proliferation rate of over-expressed aromatase MCF-7-VC3AI cells was about 1.2 times than that of the control group(P<0.01)and the proliferation rate of ZR7530-VC3AI cells was about 1.5 times than that of the control group(P<0.01). However,letrozole inhibited the proliferation induced by testosterone in a dose-dependent manner.Under the effect of letrozole at 10 μmol/L,the proliferation rate of over-expressed aromatase MCF7-VC3AI cells was 80% of the control group(P<0.05),while the prolifer-ation rate of over-expressed aromatase ZR7530-VC3AI cells was 68% of the control group(P<0.05).Conclusions:The successful estab-lishment of cell models that can detect letrozole-induced apoptosis provides an important foundation for further investigating the mechanism of letrozole.