成球培养和以cr-1为标志物分离鉴定食管癌干细胞的分析-analysis of bulking culture and identification of esophageal cancer stem cells with cr - 1 as marker.docxVIP

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成球培养和以cr-1为标志物分离鉴定食管癌干细胞的分析-analysis of bulking culture and identification of esophageal cancer stem cells with cr - 1 as marker.docx

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成球培养和以cr-1为标志物分离鉴定食管癌干细胞的分析-analysis of bulking culture and identification of esophageal cancer stem cells with cr - 1 as marker

PAGE PAGE 5 中图分类号：R737.31 文献标识码：A 英文摘要 Isolation and Characterization of Cancer Stem Cells in Esophageal Sequamous Cell Carcinoma by Tumorsphere Formation and Using Cripto-1 as a Surface Marker Abstract Objective To isolate / enrich and characterize the cancer stem cells (CSCs) in esophageal sequamous carcinoma cells (ESCCs) cell lines using methods of tumor sphere formation and Fluorescence-activated cell sorting with Cripto-1(CR-1) as a cell surface marker. Methods 1. EC109 cells were cultivated in poly-Hema coated dishes or commercial low adhesion plates with serum-free stem cell medium (DMEM/F12 1:1) supplemented with 1× B27 and the cancer stem cells reforming into floating spheres were isolated. The expression of stemness relative transcriptional factors of the spheres was measured by qRT-PCR. Consecutive passage and plate clone formation was employed to estimate the self-renewal capacity. Immunofluorescence cytochemistry was used to detect the changes of stemness relative transcriptional factors and epithelial markers expressed in spheres before and after differentiation. Xenograft assay in nude mice was performed to estimate tumorigenesis in vivo. 2. To determine the HYPERLINK /search?q=possibilityamp;keyfrom=E2Ctranslation possibility of CR-1 as a marker of ESCC CSCs, the relationship between the CR-1 expression and clinical HYPERLINK /search?q=pathologicalamp;keyfrom=E2Ctranslation pathological HYPERLINK /w/parameters/ parameters was HYPERLINK /search?q=ascertainamp;keyfrom=E2Ctranslation ascertained in clinical samples of ESCCs by Immunohistochemistry (IHC), and the survival curve was drawn. Using HYPERLINK /search?q=flowamp;keyfrom=E2Ctranslation flow HYPERLINK /w/cytometry/ cytometry, CR-1HIGH and CR-1LOW subpopulations were sorted out in EC-109 and TE-1cells. The expression of stemness genes and abilities of clony formation and invasion were compared between the two subpopulations. The tumorigenesis in nude mice was observed with or w