乳腺癌细胞CD44^+CD24^-/low亚群化疗药物外排作用研究  

Efflux function research of CD44^+CD24^-/low subsets in breast cancer cells for chemotherapy drugs

作　　者：孙洪光[1] 孝飞飞[2] 贾中明[1] 王希龙[1] 张国强[1] 杨振林[1] SUN Hong-guang;XIAO Fei-fei;JIA Zhong-ming(Department of Thyroid and Breast Surgery,Affiliated Hospital of Binzhou Medical University,Binzhou 256603,China)

机构地区：[1]滨州医学院附属医院甲状腺乳腺外科,256603 [2]滨州医学院附属医院儿科,256603

出　　处：《中国实用医药》2020年第36期199-202,共4页China Practical Medicine

基　　金：滨州医学院科研计划与科研启动基金资助项目(项目编号:BY2014KJ36)。

摘　　要：目的研究乳腺癌细胞CD44^+CD24^-/low细胞亚群对化疗药物的外排作用。方法培养多药耐药MCF-7/ADR细胞株,应用免疫磁珠法(MACS)分选CD44^+CD24^-/low亚群,流式细胞仪检测分选前后CD44^+CD24^-/low细胞比例,四甲基偶氮唑盐微量酶反应比色法(MTT法)检测化疗药物敏感程度,激光共聚焦显微镜下观察胞内阿霉素蓄积,流式细胞仪检测P-糖蛋白(P-gp)表达水平。分析MACS分选CD44^+CD24^-/low亚群结果,分选前后CD44^+CD24^-/low细胞比例,CD44^+CD24^-/low亚群对化疗药物的敏感性,CD44^+CD24^-/low亚群胞内化疗药物蓄积,CD44^+CD24^-/low细胞亚群的P-gp表达水平。结果MACS分选前后分别计数细胞数量,被分选出来的CD44^+CD24^-/low细胞比例约为6%。MCF-7/ADR细胞中CD44^+CD24^-/low细胞比例为6.21%,与分选所得CD44^+CD24^-/low细胞比例相符。经流式细胞仪检测,分选所得CD44^+CD24^-/low亚群中CD44^+CD24^-/low细胞比例约为93.85%,达到进一步相关试验要求。CD44^+CD24^-/low亚群的半数杀伤浓度(IC50)为(17.37±0.46)mg/ml,MCF-7/ADR细胞的IC50为(15.84±0.66)mg/ml,比较差异具有统计学意义(P<0.05)。激光共聚焦显微镜下观察发现,MCF-7/ADR细胞及分选所得CD44^+CD24^-/low亚群均具有较强的药物外排作用,阿霉素多集中于细胞核膜表面内侧,在细胞核中心阿霉素蓄积较少。MCF-7/ADR细胞较CD44^+CD24^-/low亚群胞内蓄积更高浓度阿霉素,荧光强度分别为(571.67±11.15)和(518.67±11.93),比较差异有统计学意义(P<0.05)。CD44^+CD24^-/low细胞亚群P-gp表达荧光强度为(114906.70±2560.19),MCF-7/ADR细胞P-gp表达荧光强度为(101177.10±2171.86),比较差异有统计学意义(P<0.05)。结论经MACS分选所得CD44^+CD24^-/low细胞亚群有更强的药物外排能力,高表达P-gp蛋白,可能是引起乳腺癌多药耐药的重要原因。Objective To study the efflux function of CD44^+CD24^-/low subsets in breast cancer cells for chemotherapy drugs.Methods The multidrug resistant MCF-7/ADR cell line was cultivated,and magnetic activated cell sorting(MACS)to sort CD44^+CD24^-/low subsets,and detect the ratio of CD44^+CD24^-/low cells before and after sorting by flow cytometry.methyl thiazolyl tetrazolium(MTT)to detect the sensitivity of chemotherapy drugs,fluorescence intensity to observe intracellular doxorubicin accumulation,flow cytometry to detect P-glycoprotein(P-gp)expression level.The results of sorting CD44^+CD24^-/low subsets by MACS,the proportion of CD44^+CD24^-/low cells before and after sorting,the sensitivity of CD44^+CD24^-/low subsets to chemotherapeutic drugs,the accumulation of chemotherapeutic drugs in CD44^+CD24^-/low subsets,and P-gp expression in CD44^+CD24^-/low subsets were analyzed.Results The number of cells was counted before and after MACS sorting,and the ratio of CD44^+CD24^-/low cells was about 6%.The ratio of CD44^+CD24^-/low cells in MCF-7/ADR cells was 6.21%,which was consistent with the ratio of CD44^+CD24^-/low cells obtained by sorting.According to flow cytometry,the ratio of CD44^+CD24^-/low cells in the sorted CD44^+CD24^-/low subsets was about 93.85%,which met the requirements of further relevant experiments.The 50%inhibition concentration(IC50)of CD44^+CD24^-/low subsets was(17.37±0.46)mg/ml,and the IC50 of MCF-7/ADR cells was(15.84±0.66)mg/ml,and the difference was statistically significant(P<0.05).Observation under a laser confocal microscope revealed that MCF-7/ADR cells and the sorted CD44^+CD24^-/low subsets all had a strong drug efflux effect.Doxorubicin is mostly concentrated on the inner surface of the nuclear membrane,and doxorubicin accumulates less in the center of the nucleus.MCF-7/ADR cells accumulated a higher concentration of doxorubicin than the CD44^+CD24^-/low subsets,and the fluorescence intensity was(571.67±11.15)and(518.67±11.93),respectively,the difference was statistically signific

关 键 词：乳腺癌 肿瘤干细胞 多药耐药 免疫磁珠法 化疗药物 外排作用 

分 类 号：R737.9[医药卫生—肿瘤]