无血清分离人结直肠癌细胞的体外培养形态及标志物表达特征

Characteristics of Morphology and Stem Cell Related Markers of Colorectal Cancer Cells in Vitro in Serum-free Medium

目的探讨人结直肠肿瘤干细胞在体外分化过程中细胞形态和干细胞相关标志物CD133的表达变化,为进一步研究结直肠肿瘤干细胞分化走向提供实验依据。方法取来源于人结直肠癌的细胞系HCT116,无血清培养分离出CD133+细胞,加血清诱导分化,相差显微镜下观察其形态变化;在未分化状态下无血清培养第7天和14天与血清诱导分化后收集细胞,利用流式细胞仪检测干细胞标志物CD133的表达量,采用激光共聚焦检测CD133表面标记分子的表达。结果 1)细胞形态:无血清培养分离的CD133+细胞,在生长过程中聚集成规则的细胞球,血清诱导后即贴壁生长,贴壁形态与同来源细胞形态一致,且再次无血清悬浮培养后聚集成球稳定生长。2)标志物变化:结直肠肿瘤干细胞未分化时CD133表面标记分子高表达,流式细胞仪检测未分化细胞CD133第7天表达率为(20.4±0.52)%,第14天表达率为(78.5±2.80)%,分化后表达率为(0.50±0.17)%。结论细胞形态和标志物表达改变均表明高表达CD133+的HCT116结直肠癌肿瘤干细胞可定向分化为同源的结直肠癌细胞,CD133+细胞经血清诱导后表达下调而使细胞失去干细胞特性。

Objective To pursue the changes of cell morphology,expression of stem cell related markers of colorectal cancer stem cells after differentiation in vitro.Methods Tumor stem cells of the line CD1 33 ＋were obtained from colorectal cancer cell line HCT1 1 6 cells were seeded in the serum free medium（SFM）supplemented with cell growth factors.Then cultured to differentiate in medium containing 1 0% fetal bovine serum.The morphology of the cells was observed under phase con-trast microscope.Cells were collected respectively before differentiation and 7,1 4 days after the differentiation.The cell sur-face markers such as CD1 33 was investigated by flow cytometry.Results 1 ）HCT1 1 6 cells were formed cancer cell spheres in SFM,Sphere cells are able to produce a heterogeneous cell population that can produce spheres.Cancer cell spheres were induced to differentiate by serum supplemented medium,these differentiated cell were adherence growth and kept the same morphology with the parental cells.2）These cancer cell spheres were founded expressing markers such as CD1 33 was higher than differentiated cells.The expression rates of CD1 33 was （20. 4 ±0. 52）% in the 7 days ,（78. 5 ±2. 80）% in the 1 4 days,and （0. 50 ±0. 1 7）% of the differentiated cells（P〈0. 05）Conclusion The changes of morphology and cell marker expression in the high-expression CD1 33 ＋CRC cells were oriented differentiated into the same origin CRC cells,and the CD1 33 ＋cells were losing stem cell ability through serum-induced.