MDA-MB-231乳腺癌细胞两种低氧模型的建立与比较分析  

Establishment and comparative analysis of 2 hypoxia models of human breast cancer MDA-MB-231 cells

作　　者：方天星 倪玉芳 赖德平 曾凡才 FANG Tian-xing;NI Yu-fang;LAI De-ping;ZENG Fan-cai(Laboratory of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Southwest Medical University,Luzhou 646000,China;School of Public Health,Southwest Medical University,Luzhou 646000,China)

机构地区：[1]西南医科大学基础医学院生物化学与分子生物学教研室,四川泸州646000 [2]西南医科大学公共卫生学院,四川泸州646000

出　　处：《中国病理生理杂志》2020年第2期244-251,共8页Chinese Journal of Pathophysiology

基　　金：四川省科技厅自然科学研究计划项目(No.LY100; No.2013SZZ001);四川省大学生创新创业训练计划项目(No.S201910719088)

摘　　要：目的:由于实体瘤内的肿瘤细胞常处于低氧环境,本实验拟探究两种常用低氧模型对乳腺癌细胞影响的差异。方法:以不同浓度(0~300μmol/L)CoCl2处理人乳腺癌MDA-MB-231细胞不同时间(24 h、48 h和72 h)建立化学低氧模型,以不同程度低氧(1%、2%和5%O2)处理MDA-MB-231细胞不同时间(4 h、16 h和24 h)建立物理低氧模型。采用CCK-8法检测细胞活力,Western blot检测低氧诱导因子1α(HIF-1α)、Bcl-2及基质金属蛋白酶2(MMP-2)的蛋白表达;流式细胞术检测细胞凋亡;Transwell和划痕实验检测细胞的侵袭及迁移能力。结果:综合不同时间和浓度的HIF-1α蛋白表达结果,最终以100μmol/L CoCl2处理24 h建立化学低氧模型,以2%O 2处理24 h建立物理低氧模型;物理低氧细胞的HIF-1α蛋白表达明显高于化学低氧,且物理低氧细胞处于复氧环境后HIF-1α蛋白快速降解;物理低氧细胞凋亡增加,抗凋亡蛋白Bcl-2表达降低,而化学低氧细胞无明显变化;两种低氧细胞的MMP-2蛋白表达均升高,且化学低氧细胞的侵袭及迁移能力增强。结论:两种低氧细胞模型构建成功,并且两种低氧细胞在HIF-1α蛋白表达与稳定性、细胞活力、凋亡、侵袭及迁移等方面均存在差异。这些差异可为低氧模型的选择和研究低氧环境下的肿瘤细胞特性提供参考。AIM:To explore the differences in the effects of 2 representative hypoxia models on human breast cancer MDA-MB-231 cells.METHODS:To establish a chemical hypoxia model,MDA-MB-231 cells were treated with CoCl 2 at different concentrations(0~300μmol/L)for different time(24 h,48 h and 72 h).On the other hand,MDA-MB-231 cells were exposed to hypoxia with different volume fractions(1%,2%and 5%)of oxygen for different time(4 h,16 h and 24 h)to establish a physical hypoxia model.The viability of the cells in the 2 hypoxia models was measured by CCK-8 assay.The protein levels of hypoxia-inducible factor-1α(HIF-1α),Bcl-2 and matrix metalloproteinase-2(MMP-2)were determined by Western blot.The apoptosis of the cells was analyzed by flow cytometry.The cell invasion and migration were examined by Transwell assay and wound-healing assay,respectively.RESULTS:The HIF-1αwas expressed in time-and dose-dependent manners.These results indicated that treatment of MDA-MB-231 cells with CoCl 2 at 100μmol/L for 24 h served as the final chemical hypoxia model,and exposure of MDA-MB-231 cells to hypoxia with 2%oxygen for 24 h served as the final physical hypoxia model.Compared with the cells with chemical hypoxia,the protein le-vel of HIF-1αwas significantly increased in the cells with physical hypoxia.In addition,reoxygenation from hypoxia caused a rapidly degraded HIF-1αexpression level in physical hypoxia model.Flow cytometry results showed that apoptosis was increased and the protein expression level of anti-apoptotic Bcl-2 was decreased in the cells with physical hypoxia,while no significant change was observed in the cells with chemical hypoxia.The protein expression level of MMP-2 in both hypoxia models was increased,and the invasion and migration capabilities of the cells with chemical hypoxia were enhanced.CONCLUSION:Two hypoxia models were successfully constructed.There were differences in the expression and stability of HIF-1αprotein,and the viability,apoptosis,invasion and migration of the cells in the 2 different hypo

关 键 词：低氧 低氧诱导因子1Α 细胞凋亡 细胞活力 乳腺癌 

分 类 号：R737.9[医药卫生—肿瘤] R730.23[医药卫生—临床医学]