内皮细胞对结肠癌细胞系SW480中CD133^+细胞表达VEGF的影响

Effect of endothelial cells on VEGF expression in CD133^+ cells from SW480 cells

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 the colon cancer cell line SW480 and investigate the effect of endothelial cells on VEGF expression in these cells.Methods Serum-free medium under floating culture system and magnetic activated cell sorting techniques were used to enrich or separate CD133＋ cells from SW480 cells.The biological characteristics of CD133＋ cells were estimated by MTT method and colony formation assay.The expression of CD133＋ and VEGF of the CD133＋ cell-formed tumor spheroids and SW480 cells were detected by indirect immunofluorescence staining.The SW480 cells and CD133＋ cells were cultured in 6 different conditions：SW480 cells were cultured in serum-supplied medium （SSM）,or serum-free medium （SFM）;CD133＋ cells were cultured in SFM;SW480 cells were cocultured with endothelial cells in SSM;SW480 cells were cocultured with in SFM;CD133＋ cells were cocultured with in SFM.The expression of CD133 and VEGF on colon cancer cells were detected by immunohistochemistry.Results There were a small number of CD133＋ cells in SW480 cells,and these cells could be continuously passaged and had a stronger ability of proliferation and colony formation than SW480 cells.The results of immunofluorescence showed SW480 cells had high expression of VEGF and low expression of CD133.However,CD133＋ cell-formed tumor spheroids had low expression of VEGF and high expression of CD133.In SW480 cells under serum-free conditions,the proportion of CD133＋ cells were gradually increased with the elapse of culture time.When those SW480 cells co-cultured with endothelial cells,the proportion was increased significantly （P0.05）.After 7 days’ continuous culture,the expression of VEGF of CD133＋ cells had no obvious change,whereas CD133＋ cells co-culture with endothelial cells showed increased expression of VEGF significantly.Under serum-free culture condition,the expression of VEGF in SW480 cells showed a decreased trend with time elapse.However,the expression was increased significantly when SW480 cells were cocultured with endothelial cells.Conclusion Endothelial cells maintain the self-renewal and undifferentiated phenotype of CD133＋ cells,which may also promote angiogenesis by inducing CD133＋ SW480 cells to express VEGF.