鼠源乳腺癌多药耐药细胞株建立及耐药机制初步研究.docVIP

鼠源乳腺癌多药耐药细胞株建立及耐药机制初步研究 　　[摘要] 目的 构建鼠源性乳腺癌5-氟尿嘧啶（5-Fu）耐药细胞株，为研究乳腺癌耐药与体内免疫微环境的关系提供细胞模型。 方法 通过体外以浓度递增的方法诱导小鼠乳腺癌4T1细胞对5-Fu产生耐药，MTS法确定其耐药性，平板克隆检测其增殖活性，实时定量和半定量PCR检测其中5-Fu代谢相关酶TS、MTHFR、TK、OPRT、DPD及药泵蛋白MDR1、MRP1的表达，通过流式细胞术检测其周期及CD44+CD24-干细胞亚群的比例。 结果 耐药细胞4T1/5-Fu与4T1细胞相比，4T1/5-Fu细胞对5-Fu、吉西他滨和顺铂耐药的耐药指数（RI）明显升高；5-Fu合成代谢相关酶TK、OPRT表达明显降低（P 0.05），代谢抑制性相关酶TS、MTHFR明显升高（P 0.05），药泵蛋白MDR1、MRP1表达明显升高（P 0.05）；流式细胞术结果显示，CD44表达明显升高，肿瘤干细胞群增多。 结论 成功构建小鼠乳腺癌多药耐药细胞株，耐药发生可能与5-Fu代谢酶类、药泵蛋白表达以及干细胞比例改变相关。 　　[关键词] 肿瘤耐药；4T1细胞；5-氟尿嘧啶；肿瘤干细胞 　　[中图分类号] R329.24 [文献标识码] A [文章编号] 1673-7210（2016）04（b）-0009-06 　　[Abstract] Objective To construct 5-fluorouracil （5-Fu） resistant murine breast cancer cell line， in order to provide cell model for study of relationship between breast cancer multidrug resistant and immune microenvironment in vitro. Methods 4T1 cells were exposed in stepwise escalatingconcentration of 5-Fu to develop the resistant cell line 4T1/5-Fu. And the chemosensitivity and proliferation of 4T1/5-Fu were determined by MTS and colony forming experiments， respectively. Furthermore， real-time RT-PCR and semi quantitative PCR were used to measure expression levels of 5-Fu related genes， such as TS， MTHFR， TK， OPRT， DPD， MDR1， MRP1. In addition， cell cycle and the proportion of CD44+CD24- stem cell subsets were evaluated by flow cytometry. Results Comparing to 4T1 cells， the resistance index of 4T1/5-Fu cells to 5-Fu， GEM， cDDP was increased. The results showed that compared with 4T1 cells， the expression levels of TK and OPRT were significantly decreased （P 0.05）， while TS， MTHFR， MDR1 and MRP1 were remarkably increased in 4T1/5-Fu cells （P 0.05）. Flow cytometry assay showed that the propotion of tumor stem cells CD44+CD24- was evidently increased in 4T1/5-Fu cells than that of 4T1 cells. Conclusion This article has successfully constructed multidrug resistant cell line 4T1/5-Fu， whose resistance may be associated with up-regulation of the 5-Fu metabolic enzymes， drug pump proteins exp