人乳腺癌MCF-7细胞中SP亚群细胞的分离及其生物学行为研究

Sorting and biological behaviors of cancer stem cells in human breast cancer MCF-7 cells

目的:了解肿瘤干细胞的生物学行为及相关的调控机制。方法:采用干细胞对Hoechst33342染料外排的特性进行MCF-7细胞系肿瘤干细胞的流式分选。并对其增殖、自我更新、分化能力等生物学行为进行研究。还利用流式细胞仪、免疫荧光技术、荧光定量PCR等技术进行了与干细胞生物学行为密切相关的Wnt信号转导通路中相关分子的表达特征研究。结果:MCF-7细胞系中包含SP亚群(side population);且SP细胞具有分化潜能,高增殖能力,及自我更新特性;而且Wnt通路的各种关键分子在该细胞系SP亚群细胞中呈活化状态。结论:MCF-7细胞系的SP亚群可以代表该细胞系的肿瘤干细胞,Wnt通路在该肿瘤细胞系肿瘤干细胞的自我更新等其它生物学行为的调控中起重要作用。

Objective：To understand the biological behaviors of cancer stem cells and associated regulation mechanisms.Methods： To sort cancer stem cells from MCF-7 cells,which we identified and sorted basing Hoechst 33342 dye exclusion of stem cells.And the subpopulation of the cells which could efflux Hoechst33342 called SP cells.Then we studied the biological behaviors of SP cells and non-SP cells,such as proliferation,differentiation,and self-renewal.And we applied flow cytometry,immunofluorescence technic,and fluorescent quantitation RT-PCR to study the key molecules of Wnt signaling pathway which is closely related to the biological behaviors of stem cells.Results： The percentage of SP in MCF-7 cells was（4.01±0.18）%;SP cells perform a higher speed proliferation rate and higher clone formation rate than non-SP cells;the re-cultured SP cells can differentiate into SP and non-SP cells;the expressions of β-catenin,c-myc and cyclinD1 were higher,phosphorylated β-catenin was lower,as compared with non-SP cells.Conclusion： The activity of Wnt signaling pathway is over-regulated in SP cells.