氧化苦参碱干预MCF-7细胞系肿瘤干细胞样细胞生物学行为的实验研究

Oxymatrine Diminished the Side Population and Inhibited Wnt/β-catenin Signaling Pathway in MCF-7 Breast Cancer Cells

目的研究中药单体氧化苦参碱(oxymatrine,OM)对人乳腺癌细胞系MCF-7肿瘤干细胞样细胞生物学行为的干预作用及分子水平作用机制。方法以MCF-7为研究对象,采用侧群分选法(side population,SP)分离富集MCF-7细胞系肿瘤干细胞样细胞(SP细胞);MTT技术检测SP细胞和非肿瘤干细胞样细胞(non-side population cells,non-SP细胞)的增殖特性;以及不同浓度顺铂(cisplatin,DDP)和OM对于不同细胞亚群的增殖干预作用;利用流式细胞技术、细胞免疫荧光技术检测细胞中β-catenin基因、蛋白表达水平。结果 (1)不同浓度OM对MCF-7细胞的不同亚群细胞增殖抑制作用不同,其中对non-SP细胞的增殖抑制作用最弱,未分选的细胞次之,对SP抑制作用最强;不同浓度DDP对non-SP的增殖抑制作用最强,未分选的细胞次之,对SP细胞抑制作用最弱;(2)浓度为0.25、0.50、1.00mg/mL的OM作用SP细胞后,其在细胞总数中所占比例分别为3.1%、1.7%、0.2%;经过OM处理过的SP细胞磷酸化β-catenin表达率为(42.62±2.62)%,与未经OM处理过SP细胞的表达率[(22.81±1.66)%]比较,差异有统计学意义(P<0.01);(3)与未经OM处理过的SP细胞比较,经过OM处理过的SP细胞中β-catenin的表达明显减少,而且特异性地分布在细胞膜下,核转位现象明显减少。结论 OM可能通过抑制Wnt/β-catenin信号转导通路的活性来干预人乳腺癌MCF-7细胞系中肿瘤干细胞样细胞的生物学行为。

Objective To observe the effects of oxymatrine（OM）in inhibiting the proliferation and percentage of cancer stem cell like cell of human breast cancer MCF-7 cells,and to study its molecular mechanism.Methods MCF-7 cells were taken as subject.Side population cells（SP）of cancer stem cell like cells rich in MCF-7 cells were isolated using side population（SP）method.The proliferation properties of SP cells and non-side population（non-SP）cells were detected by MTT assay.The proliferation and intervention of cisplatin（DDP）and OM at various concentrations were detected as well.The expression levels ofβ-catenin gene and protein were detected using flow cytometry and immunofluorescence technique.Results（1）OM in different concentrations had various inhibitions to the proliferation of subpopulations of MCF-7 cells.Of them,it had weakest inhibition on non-SP cells,weaker inhibition on unsorted cells,and strongest inhibition on SP cells.DDP in different concentrations had strongest inhibition on non-SP cells,weaker inhibition on unsorted cells,and weakest inhibition on SP cells.（2）SP cells accounted for 3.1%,1.7%,and0.2% of the total cells after OM acted at 0.25,0.50,1.00 mg/mL,respectively.The expression rate of phosphorylatedβ-catenin was 42.62%±2.62% after SP cells were acted by OM,with statistical difference as compared with the blank control group[（22.8%±1.66%）,P〈0.01].（3）Compared with SP cells without OM treatment,the expression ofβ-catenin in OM treated SP cells was obviously reduced.Besides,they were specifically distributed under the cytomembrane,with nuclear translocation obviously reduced.Conclusion OM could intervene biological behaviors of cancer stem cell like cell of MCF-7 cells possibly through Inhibiting the activation of Wnt/β-catenin signaling pathway.