Evidence for EpCAM and Cytokeratin Expressing Epithelial Cells in Normal Human and Murine Blood and Bone Marrow.
Evidence for EpCAM and Cytokeratin Expressing Epithelial Cells in Normal Human and Murine Blood and Bone Marrow.
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DOI：
10.3791/65118
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发表时间：
2023-04-21
期刊：
影响因子：
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通讯作者：
Morris R
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文献类型：
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作者：
Holtorf SM;Boyle J;Morris R
已经在癌症和其他疾病患者的血液和骨髓中鉴定出上皮细胞。然而，健康个体的血液和骨髓中是否存在正常上皮细胞还有待以一致的方式鉴定。本文介绍了一种可重复的方法，用于使用流式细胞术和免疫荧光（IF）显微镜从健康人和小鼠血液和骨髓（BM）中分离上皮细胞。首先使用上皮细胞粘附分子（EpCAM）通过流式细胞术鉴定和分离健康个体中的上皮细胞。使用免疫荧光显微镜和Krt1 - 14; mTmG转基因小鼠证实这些EpCAM阳性细胞表达角蛋白。人血液样品具有0.18% ± 0.0004的所有细胞EpCAM+（平均值的标准误差[SEM]; n = 7个生物学重复，4个实验重复）。在人BM中，3.53% ± 0.006（SEM; n = 3个生物学重复，4个实验重复）的单核细胞为EpCAM+，无论计数的细胞数量如何。在小鼠血液中，EpCAM+细胞构成0.45% ± 0.0006（SEM; n = 2个生物学重复，4个实验重复），并且在小鼠BM中，5.17% ± 0.001（SEM; n = 3个生物学重复，4个实验重复）是EpCAM+。在小鼠中，几乎所有的EpCAM+细胞免疫反应泛细胞角蛋白，如通过IF显微镜测定。使用Krt1 - 14; mTmG转基因小鼠证实了结果，与多个阴性对照相比，在正常鼠BM中存在低（每106个分析的细胞中8.6个天然GFP+细胞; 0.085%的活细胞）但显著数量（p <0.0005）的GFP+细胞，这不是随机性的结果。此外，小鼠血液中的EpCAM+细胞比CD45+细胞更异质（BM中0.58%;血液中0.13%）。这些观察结果得出结论，表达细胞角蛋白的细胞在人和鼠血液和BM的单核细胞中可重复检测。本报告展示了一种方法，组织收获，流式细胞术，免疫染色，可用于识别和确定这些泛细胞角蛋白上皮细胞在健康个体的功能。本文将提出一种可重复的方法，在正常人和小鼠的血液和骨髓中使用流式细胞术和免疫荧光显微镜上皮细胞的存在新的发现。Krt1 - 14; mTmG转基因小鼠被用作体内方法来证实这些发现。
Epithelial cells have been identified in the blood and bone marrow of patients with cancer and other diseases. However, the presence of normal epithelial cells in the blood and bone marrow of healthy individuals had yet to be identified in a consistent way. Presented here is a reproducible method for isolating epithelial cells from healthy human and murine blood and bone marrow (BM) using flow cytometry and immunofluorescence (IF) microscopy. Epithelial cells in healthy individuals were first identified and isolated via flow cytometry using epithelial cell adhesion molecule (EpCAM). These EpCAM positive cells were confirmed to express keratin using immunofluorescence microscopy and Krt1–14;mTmG transgenic mice. Human blood samples had 0.18% ± 0.0004 of all cells EpCAM+ (standard error of the mean [SEM]; n=7 biological replicates, 4 experimental replicates). In human BM, 3.53% ± 0.006 (SEM; n=3 biological replicates, 4 experimental replicates) of mononuclear cells were EpCAM+, regardless of the number of cells counted. In mouse blood, EpCAM+ cells constituted 0.45% ± 0.0006 (SEM; n=2 biological replicates, 4 experimental replicates) and in mouse BM, 5.17% ± 0.001 (SEM; n=3 biological replicates, 4 experimental replicates) were EpCAM+. In mice, virtually all the EpCAM+ cells were immunoreactive to pan-cytokeratin as determined by IF microscopy. Results were confirmed using Krt1–14;mTmG transgenic mice, with low (8.6 native GFP+ cells per 106 cells analyzed; 0.085% of viable cells), but significant numbers (p<0.0005) of GFP+ cells present in normal murine BM that were not the result of randomness compared with multiple negative controls. Further, EpCAM+ cells in mouse blood were more heterogeneous than CD45+ cells (0.58% in BM; 0.13% in blood). These observations conclude that cells expressing cytokeratin proteins are reproducibly detectable among mononuclear cells from human and murine blood and BM. This report demonstrates a method of tissue harvesting, flow cytometry, and immunostaining that can be used to identify and determine the function of these pan-cytokeratin epithelial cells in healthy individuals. This paper will present a reproducible method with new findings on the presence of epithelial cells in normal human and mouse blood and bone marrow using flow cytometry and immunofluorescence microscopy. Krt1–14;mTmG transgenic mice were used as an in vivo method to confirm these findings.