三阴性乳腺癌干细胞的富集及CD44+CD24-/low、CXCR4表达测定

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。

Enrichment of Triple Negative Breast Cancer StemCells and Detecting thePropotion of CD44+CD24-/low and CXCR4 Protein Expression

Objective:In order to acquire triple-negative breast cancer stem cells by adherent MDA-MB-231 cells were cultured inserum-free medium. In the experiment, the morphological observation of the sphere re-formation and colony formation was carried out, inaddition, the protein expression of CD44+CD24-/low and CXCR4 was detected.Methods:Human MDA-MB-231 breast cancer cell lineswere plated in stem cell conditioned culture system to generate mammospheres. Samples of mammospheres in 7-9 days were taken toevaluate the effects of different MDA-MB-231cell density on the mammosphere-formation efficiency; the proportion of CD44+CD24-/lowwas analyzed by flow cytometry, CXCR4 protein expression was tested by western blot, sphere re-formation and colony formation wereassessed by morphological observation.Results:Triple negative breast cancer mammospheres can be enriched in serum-free mediumcon- 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 ofCD44+ CD24-/low in MDA-MB-231 adherent cells and mammosheres were (38.54 ± 2.00) % VS (66.35 ± 2.06) %, P=0.003. CXCR4protein expression in mammosheres cells was higher than MDA-MB-231 adherent cells, P=0.03. Single mammosphere cell has the abilityto generate sphere again. Colony formations of mammosheres need 200 cells and adherent MDA-MB-231 need 1000 cells in soft agarmedia.Conclusion:1.Triple negative breast cancer stem cells can be enriched in serum-free medium, low cell density was beneficial to theformation of mammosheres. 2. Mammosheres had a higher CD44+CD24-/low proportion than MDA-MB-231 adherent cells. 3.CXCR4protein expression in mammosheres was higher than in MDA-MB-231 adherent cells.