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目的:研究通过5氟尿嘧啶(5-FU)对结肠癌干细胞标记物CD133表达的影响,探讨5-FU对结肠癌干细胞的影响。方法:用流式细胞仪检测CD133在结肠癌细胞株表面的表达,磁珠细胞分离的方法分离结肠癌细胞株DLD1中CD133阳性和阴性的细胞群,细胞克隆形成实验检测2群细胞的自我更新能力,新型四唑氮盐方法(MTS)检测2群细胞对5-FU敏感性的差异,qPCR方法检测5-FU处理结肠癌细胞后CD133 mRNA水平的变化。结果:结肠癌细胞株DLD1、HT29、SW480、HCT116、Lovo、RKO细胞表面CD133的表达率分别为30.20%、82.00%、0.34%、91.80%、85.30%、0.28%。DLD1细胞中以CD133为标记有2群明显的细胞,MACS方法分离后阳性细胞群中CD133为87.21%±5.33%,而阴性细胞群中阴性细胞的比例为84.30%±4.65%;CD133阳性的细胞与未分离及CD133阴性细胞相比,克隆形成能力强(46.33%±4.44%vs31.00%±2.00%,P<0.05),对5-FU的敏感性下降20%,P<0.01。在DLD1和HT29细胞中,5-FU1mg/L上调CD133 mRNA水平的表达,从1升为1.684±0.012(P<0.01)、HT29细胞从30.702±0.284升为49.379±0.460(P<0.01)。结论:与CD133阴性细胞相比CD133阳性细胞克隆形成能力强,对5-FU的敏感性下降;5-FU上调干细胞标记物CD133 mRNA水平的表达,CD133阳性的结肠癌干细胞在5-FU的治疗过程中被富集。
Objective: To investigate the effect of 5-fluorouracil (5-FU) on the expression of CD133, a marker of colon cancer stem cells, to investigate the effect of 5-FU on colon cancer stem cells. Methods: The expression of CD133 on the surface of colon cancer cell lines was detected by flow cytometry. The cell population of CD133 positive and negative colon cancer cell lines DLD1 was isolated by magnetic bead separation. The cell clone formation assay was used to detect the self-renewal of two groups of cells Ability, the new tetrazolium salt method (MTS) to detect the sensitivity of 2 groups of cells to 5-FU sensitivity, qPCR method to detect 5-FU treatment of colon cancer CD133 mRNA levels. Results: The expression rates of CD133 on colon cancer cell lines DLD1, HT29, SW480, HCT116, Lovo and RKO were 30.20%, 82.00%, 0.34%, 91.80%, 85.30% and 0.28%, respectively. In DLD1 cells, CD133 was marked with 2 groups of obvious cells. After MACS method, the percentage of CD133 positive cells was 87.21% ± 5.33% while that of negative cells was 84.30% ± 4.65%. The number of CD133 positive cells Compared with non-isolated and CD133-negative cells, the clonogenic capacity was significantly higher (46.33% ± 4.44% vs31.00% ± 2.00%, P <0.05) and the sensitivity to 5-FU was reduced by 20% (P <0.01). In DLD1 and HT29 cells, the expression of CD133 mRNA increased from 1 to 1.684 ± 0.012 (P <0.01) with the increase of 5-FU at 1 mg / L, and increased from 30.702 ± 0.284 to 49.379 ± 0.460 (P <0.01). CONCLUSION: CD133-positive cells have stronger colony formation ability and lower sensitivity to 5-FU than CD133-negative cells. 5-FU up-regulates the expression of CD133 mRNA and CD133-positive colon cancer stem cells in the treatment of 5-FU The process is enriched.