Mammary epithelial cell subpopulations from control and ovariectomized mice. Mus musculus

To study the effect of pregnancy on mouse mammary epithelial subpopulations, epthelial cells derived from control or ovariectomized adult mice were isolated using fluorescence-activated cell sorting. After elimination of haematopoietic and endothelial cells, two distinct epithelial subpopulations were sorted using antibodies against CD29 and CD24. Based on the immunohistochemical phenotype, and in vivo and in vitro functional assays, these subpopulations were identified as mammary stem cell enriched (CD29hiCD24+) and luminal (CD29loCD24+) respectively (ref: Shackleton et al, Nature 2006). Microarray profiling was used to compare gene expression profiles of the two subpopulations isolated from ovariectomized and control mice. Overall design: For each of two biological replicates, 6 FVB/NJ mice were ovariectomized at 8 weeks of age. 4 weeks after ovariectomy (Ovx) mammary gland from control or Ovx animals were collected and digested to obtain a single cell suspension. CD45-CD31-TER119- cells were then sorted based on the expression of cell surface markers CD24 and CD29. Similarly for two pools of control mice. There were also 4 technical replicates, to make 12 BeadChips in total.

为探究妊娠对小鼠乳腺上皮亚群的影响，本研究通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）分离对照组与去卵巢成年小鼠的上皮细胞。在去除造血细胞与内皮细胞后，利用针对细胞表面标记CD29与CD24的抗体分选得到两种特征明确的上皮亚群。基于免疫组化表型及体内外功能实验结果，这两种亚群分别被鉴定为乳腺干细胞富集型（CD29hiCD24+）与管腔型（CD29loCD24+）亚群（参考文献：Shackleton 等，《自然》，2006年）。本研究采用基因芯片表达谱分析，比较从去卵巢小鼠与对照组小鼠中分离得到的两种上皮亚群的基因表达谱。实验设计概述：两组生物学重复中，各有6只FVB/NJ小鼠于8周龄时接受去卵巢手术。去卵巢（Ovx）术后4周，收集对照组与去卵巢小鼠的乳腺组织并消化制备单细胞悬液。随后基于细胞表面标记CD24与CD29的表达水平，分选得到CD45-CD31-TER119-细胞。对照组小鼠样本亦设置两组平行混合样本。此外本研究设置4次技术重复，总计使用12张BeadChips。