目的从结肠癌细胞系SW480中分离结肠癌干细胞,并检测内皮细胞对这些细胞表达血管内皮生长因子(vascular endothelial growth factor,VEGF)的影响。方法分别利用免疫磁珠分选技术和无血清培养法富集CD133+细胞,利用MTT法和平板克隆形成实验检测其生物学特性;采用细胞免疫荧光法检测SW480细胞和CD133+细胞中CD133及VEGF的表达;将SW480细胞或磁珠分选CD133+细胞分别在6种培养条件下进行培养:SW480细胞在含血清培养基(serum-supplied medium,SSM)中培养、SW480细胞在无血清培养基(serum-freemedium,SFM)中培养、磁珠分选CD133+细胞在SFM中培养、SW480细胞与内皮细胞在SSM中共培养、SW480细胞与内皮细胞在SFM共培养、磁珠分选CD133+细胞与内皮细胞在SFM共培养,利用免疫组织化学法检测不同培养条件下结肠癌细胞中CD133及VEGF的表达情况。结果 SW480细胞系中存在少量的CD133+细胞,这些细胞能够连续传代并且表现出更强的增殖潜力以及克隆形成能力。经细胞免疫荧光法检测发现SW480细胞高表达VEGF,低表达CD133,而免疫磁珠分选的CD133+细胞所形成的细胞球低表达VEGF,高表达CD133。无血清培养条件下的SW480细胞随着时间的延长CD133表达率逐渐增加,添加内皮细胞诱导后无血清培养的SW480细胞与前者相比CD133阳性率显著增加(P<0.05)。经过1周的连续培养后,CD133+细胞表达VEGF的比例没有变化,添加内皮细胞诱导1周后,CD133+细胞表达VEGF的比例显著增加(P<0.01)。在无血清培养条件下,SW480细胞表达VEGF的比例随着时间的延长有所降低,但是添加内皮细胞诱导后无血清培养1周的细胞与无内皮诱导培养条件下的细胞相比VEGF表达增加(P<0.01)。结论内皮细胞能够促进CD133+细胞的自我更新维持其未分化状态,并且内皮细胞能够通过诱导CD133+细胞表达VEGF来促进血管的生成。 Objective To isolate colon cancer stem cells from human colon cancer cell line SW480 and detect the effect of endothelial cells on the expression of vascular endothelial growth factor (VEGF) in these cells. Methods CD133 + cells were enriched by immunomagnetic bead sorting and serum - free culture respectively. The biological characteristics of CD133 + cells were detected by MTT assay and plate clone formation assay. The expressions of CD133 and VEGF in SW480 cells and CD133 + cells were detected by immunofluorescence ; SW480 cells or magnetic beads sorting CD133 + cells were cultured in six culture conditions: SW480 cells were cultured in serum-supplied medium (SSM), SW480 cells in serum-free medium , SFM). Magnetic beads were used to culture CD133 + cells in SFM. SW480 cells were co-cultured with endothelial cells in SSM. SW480 cells were co-cultured with endothelial cells in SFM. Magnetic separation of CD133 + cells and endothelial cells were performed in SFM The expression of CD133 and VEGF in colorectal cancer cells under different culture conditions was detected by immunohistochemistry. Results There was a small amount of CD133 + cells in the SW480 cell line. These cells were able to pass on continuously and showed stronger proliferative potential and clonogenic capacity. The results of immunofluorescence showed that SW480 cells highly expressed VEGF and CD133 was low, whereas the immunospot-sorted CD133 + cells expressed low expression of VEGF and high expression of CD133. Compared with the former, the positive rate of CD133 in SW480 cells in serum-free culture increased gradually with the increase of time, and the positive rate of CD133 in serum-free SW480 cells increased significantly (P <0.05). The ratio of VEGF expression in CD133 + cells did not change after one week of continuous culture, and the proportion of VEGF expression in CD133 + cells increased significantly (P <0.01) after 1 week induction of endothelial cells. In serum-free culture, the percentage of VEGF expression in SW480 cells decreased over time, but VEGF expression was increased in cells cultured in serum-free medium for 1 week after addition of endothelial cells compared to cells in absence of endothelial induction culture P <0.01). Conclusions Endothelial cells promote the self-renewal of CD133 + cells to maintain their undifferentiated state. Endothelial cells promote the formation of blood vessels by inducing CD133 + cells to express VEGF.