乳腺癌干细胞富集与鉴定的初步研究

目的:通过从MCF-7、ZR-75-1、MDA-MB-231乳腺癌细胞系中培养富集及鉴定乳腺癌干细胞(breast cancer stem cell,BCSC)，寻找培养与富集乳腺癌干细胞的方法。方法:贴壁培养MCF-7、ZR-75-1、MDA-MB-231细胞系，倒置显微镜观察各细胞形态；流式细胞仪分别分选收集CD44-CD24-、CD44-CD24+、CD44+CD24-及 CD44+CD24+ 细胞，其中CD44+CD24-为乳腺癌干细胞，其余三类为对照组；MTT法计数细胞，绘制MCF-7、ZR-75-1、MDA-MB-231细胞系生长曲线；MCF-7细胞系进行无血清悬浮培养1个周期，流式细胞仪检测分子表面标记物CD44+CD24-含量，贴壁培养的CD44+CD24-乳腺癌干细胞为对照组；将分选的MCF-7（CD44+CD24-）和分选的其余MCF-7细胞（非CD44+CD24-）进行干性成球实验，鉴定CD44+CD24-干性表达。结果:MCF-7、MDA-MB-231细胞系富含表面标志物CD44-CD24-的乳腺癌细胞；ZR-75-1细胞系富含分子表面标志物CD44+CD24+的乳腺癌细胞；生长曲线显示MCF-7、ZR-75-1、MDA-MB-231均呈持续增长，MDA-MB-231细胞生长较MCF-7、ZR-75-1细胞快；通过无血清悬浮培养CD44+CD24-乳腺癌干细胞由19.4%富集到88.9%；成球实验中CD44+CD24-表型细胞成球数量较分选的其余MCF-7细胞（非CD44+CD24-表型）明显增多,成球率分别为（36.5±1.7）%，（1.1±0.5）%。结论:流式细胞仪可成功分选出分子表面标志物为CD44+CD24-的乳腺癌干细胞；CD44+CD24-可能不是乳腺癌干细胞唯一的表面标志物；MDA-MB-231细胞系较MCF-7、ZR-75-1细胞系生长快；无血清悬浮培养法可简便、高效地富集乳腺癌干细胞；CD44+CD24-乳腺癌干细胞干性表达较强。

Preliminary study on the accumulation and identification of breast cancer stem cells

Objective:A method for culturing and enriching breast cancer stem cells was discovered by enriching and identifying breast cancer stem cells from MCF-7,ZR-75-1,and MDA-MB-231 breast cancer cell lines.Methods:MCF-7,ZR-75-1 and MDA-MB-231 cell lines were subjected to adherent culture,and the morphology of each cell was observed by an inverted microscope.CD44-CD24-,CD44-CD24+,CD44+CD24- and CD44+CD24+ cells were collected by flow cytometry,in which CD44+CD24- were breast cancer stem cells,and the other three groups were the control group.Cells were counted by MTT method,and the growth curve of MCF-7,ZR-75-1 and MDA-MB-231 cell lines was drawn.MCF-7 cell line was subjected to serum-free suspension culture for 1 cycle,and the content of molecular surface marker CD44+CD24- was detected by flow cytometry.The inherently cultured CD44+CD24-breast cancer stem cells were used as the control group.The sorted MCF-7 cells(CD44+CD24-) and the sorted remaining MCF-7 cells(the sorted remaining MCF-7 cells) were subjected to stemness sphere-forming assays to identify CD44+CD24-stemness expression.Results:MCF-7 and MDA-MB-231 cell lines were rich in breast cancer cells of surface marker CD44-CD24-.ZR-75-1 cell line were rich in breast cancer cells of surface marker CD44+CD24+.Growth curve showed that MCF-7 ZR-75-1 and MDA-MB-231 cells took on continuous increase,and MDA-MB-231 cells grew faster than MCF-7 and ZR-75-1 cells.CD44+CD24-breast cancer stem cells were enriched from 19.4% to 88.9% by serum-free suspension culture.The number of CD44+CD24- phenotype cells in the sphere-forming assay was significantly higher than that of the sorted remaining MCF-7 cells,with the sphere-forming rate of (36.5±1.7)%，（1.1±0.5）%.Conclusion:Flow cytometry can successfully sort out breast cancer stem cells with molecular surface marker CD44+CD24-.Different subtypes of breast cancer vary in gene expression,invasion,metastasis,hormone sensitivity and prognosis.CD44+CD24- may not be the only surface marker of breast cancer stem cells.MDA-MB-231 cell line grows faster than MCF-7,ZR-75-1 cell lines.Serum-free suspension culture method can enrich breast cancer stem cells easily and efficiently.CD44+CD24- breast cancer stem cells have stronger stemness expression.