三阴性乳腺癌干细胞的富集及CD44+CD24-/low、CXCR4表达测定

Enrichment of Triple Negative Breast Cancer Stem Cells and Detecting the Propotion of CD44+CD24-/low and CXCR4 Protein Expression

目的:探讨MDA-MB-231细胞经无血清培养富集三阴性乳腺癌干细胞,观察再成球、集落形成及CD44+CD24-/low、CXCR4表达。方法:将MDA-MB-231乳腺癌细胞进行微球体培养,取培养第7-9天的微球体,判断干细胞富集的程度;比较不同细胞浓度对癌球细胞成球率影响;流式细胞仪测定CD44+CD24-/low含量;Western blot法分析CXCR4蛋白表达;单个癌球细胞再成球能力;观察癌球与贴壁细胞集落形成。结果:1).在含20 ng/m L EGF,10 ng/m L b FGF,2%b27无血清培养基中可培养三阴性乳腺癌癌球,1×104/m L、2×104/m L、3×104/m L、4×104/m L、5×104/m L细胞浓度癌球细胞成球率分别为(5.61±0.02)%、(3.23±0.54)%、(2.28±0.48)%、(1.05±0.13)%、(0.91±0.01)%,组间比较差异有统计学意义P值均<0.05。2).贴壁MDA-MB-231细胞与癌球细胞CD44+CD24-/low含量分别为(38.54±2.00)%VS(66.35±2.06)%,差异有统计学意义P=0.003。3).癌球细胞CXCR4蛋白表达高于贴壁MDA-MB-231细胞,灰度扫描分析差异有统计学意义,P=0.03。4).单个癌球细胞具有再成球能力。5).软琼脂糖集落形成能力癌球需200个细胞即可见集落形成,而贴壁细胞需1 000个MDA-MB-231细胞。结论:1.通过无血清培养可以富集三阴性乳腺癌干细胞,低细胞密度有利于癌球形成。2.癌球中CD44+CD24-/low含量高于贴壁MDA-MB-231细胞。3.CXCR4在癌球中表达高于贴壁MDA-MB-231细胞。

Objective：In order to acquire triple-negative breast cancer stem cells by adherent MDA-MB-231 cells were cultured in serum-free medium.In the experiment,the morphological observation of the sphere re-formation and colony formation was carried out,in addition,the protein expression of CD44＋CD24-/low and CXCR4 was detected.Methods：Human MDA-MB-231 breast cancer cell lines were plated in stem cell conditioned culture system to generate mammospheres.Samples of mammospheres in 7-9 days were taken to evaluate the effects of different MDA-MB-231 cell density on the mammosphere-formation efficiency; the proportion of CD44＋CD24-/low was analyzed by flow cytometry,CXCR4 protein expression was tested by western blot,sphere re-formation and colony formation were assessed by morphological observation.Results：Triple negative breast cancer mammospheres can be enriched in serum-free medium con-taining 20 ng/mL EGF,10 ng/mL bFGF and 2% b27.The mammosphere formation efficiency was （5.61± 0.02） %,（3.23± 0.54） %,（2.28± 0.48） %,（1.05± 0.13） % and （0.91± 0.01） % when the MDA-MB-231 cell culturing density was 1 × 104/mL,2× 104/mL,3×104/mL,4× 104/mL and 5× 104/mL,respectivel.There was a significantly difference among these groups （P〈0.05）.The proportion of CD44＋ CD24-/low in MDA-MB-231 adherent cells and mammosheres were （38.54 ± 2.00） % VS （66.35 ± 2.06） %,P=0.003.CXCR4 protein expression in mammosheres cells was higher than MDA-MB-231 adherent cells,P=0.03.Single mammosphere cell has the ability to generate sphere again.Colony formations ofmammosheres need 200 cells and adherent MDA-MB-231 need 1000 cells in soft agarmedia.Conclusions：1.Triple negative breast cancer stem cells can be enriched in serum-free medium,low cell density was beneficial to the formation of mammosheres.2.Mammosheres had a higher CD44＋CD24-/low proportion than MDA-MB-231 adherent cells.3.CXCR4 protein expression in mammosheres was higher than in MDA-MB-231 adherent cells.