人乳腺癌mcf-7他莫昔芬耐受细胞株emt表型的 - 第三军医大学学报.docVIP

人乳腺癌MCF-7他莫昔芬耐受细胞株EMT表型的鉴定及其机制的初步研究 袁杰，杨丽2陈茂山1，朱庆1，成宏3，杨光伦1（400016 重庆，重庆医科大学附属第一医院内分泌乳腺外科临床检验诊断学教育部重点实验室2 445000 湖北，恩施土家族苗族自治州中心医院乳腺外科3）[摘要] 观察乳腺癌细胞MCF-7发生他莫昔芬耐受后细胞形态的变化及上皮-间质转化（epithelial-mesenchymal transition，EMT）现象，并初步研究其机制。 免疫荧光染色观察乳腺癌细胞MCF-7及其他莫昔芬耐药株MCF-7R细胞的形态；qRT-PCR、Western blot 检测两种细胞EMT相关标志蛋白的表达； Transwell法检测两种细胞的迁移能力；分别抑制PI3K-A及MAPK/Erk信号通路，检测MCF-7R细胞的迁移能力及EMT表型的变化。MCF-7细胞间的联系紧密，发生他莫昔芬耐受后，细胞间隙明显增大；MCF-7细胞发生他莫昔芬耐受后迁移能力明显增强，并发现E-钙粘素(E-cadherin)分布从细胞膜向胞质转移，波形蛋白（vimentin）及纤维黏连蛋白（fibronectin）表达水平明显升高（P＜0.05）；在耐药细胞中PI3K-Akt及MAPK/Erk信号通路均处于异常活化状态，抑制PI3K-Akt信号通路能明显抑制细胞的迁移能力同时能使波形蛋白和纤维黏连蛋白表达水平降低（P＜0.05）。乳腺癌MCF-7细胞发生他莫昔芬耐受后其迁移能力增强可能与其发生EMT样表型有关。[关键词] 乳腺癌；他莫昔芬耐受 [中图法分类号] R737.9dentification and the preliminary research on the mechanism of epithelial-mesenchymal transition phenotype in tamoxifen-resistant MCF-7 cells Yuan Jie1, Yang Li2, Chen Maoshan1, Zhu Qing1, Cheng Hong3, Liu Manran2, Yang Guanglun1 1(Department of Endocrine and Breast Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, 2Department of Laboratory Medical Diagnosis, the Ministry of Education Key Laboratory of Laboratory Medical Diagnosis of Chongqing Medical University, Chongqing, 400016, 3Department of Endocrine Breast Surgery, the Central Hospital of Enshi Autonomous Prefecture, Hubei, 445000, China) [Abstract] Objective To investigate the changes of the cell morphology and the expression of epithelial-mesenchymal transition (EMT) marker proteins after MCF-7 cells acquired tamoxifen resistance. Methods The cell morphology was revealed through immunofluorescence. The expression of EMT marker proteins was detected by quantitative real-time PCR (qRT-PCR) and Western blotting. Moreover, Transwell assay was used to detect the difference of cell migration. PI3K-Akt and MAPK/Erk signaling pathway were evaluated in MCF-7 cells and the tamoxifen-resistant subclones MCF-7R cells, the migration and the expression of EMT marker proteins were reevaluated after inhibi