目的 观察低氧微环境对结肠癌细胞增殖、侵袭及自噬的影响,探讨低氧对结肠癌细胞生存及与自噬的关系.方法 免疫磁珠分选CD133+细胞;Boyden小室实验研究结肠癌细胞的侵袭;透射电子显微镜(TEM)观察细胞的超微结构;MDC荧光检测自噬囊泡;联合运用流式细胞仪和锥虫蓝染色研究自噬与细胞增殖的关系.结果 Boyden小室实验中低氧微环境增加结肠癌细胞SW480进入下室的细胞数量;在实验组低氧的微环境中SW480细胞的MDC荧光强度由20.53±0.64降低至17.00±0.20,而CD133+细胞的MDC荧光强度由19.89±0.58增加至33.13±1.95(P＜0.05).锥虫蓝染色实验显示,与对照组相比,低氧微环境中SW480活细胞率随着时间的延长而降低(P＜0.05):但CD133+活细胞率无显著改变.结论 低氧可增加SW480细胞的侵袭性,细胞自噬水平下降,活细胞率降低;CD133+细胞在低氧微环境中自噬的发生增加,活细胞率基本不变.%Objective To observe the effects of hypoxic microenvironment on the proliferation, invasion and autophagy of colon cancer cells and to investigate the relationship between survival and autophagy of colon cancer cells under hypoxic conditions. Methods CD 133 + cancer stem cells (cancer stem cells, CSCs) were purified using MACS (magnetic activated cell-sorting system) CD 133 + cell isolation kit. The invasion of colon cells was measured by Boyden chamber. The ultrastructure was observed using transmission electron microscope (TEM). The autophagy vesicles were detected by monodansylcadaverin ( MDC) fluorescence. The relationship between autophagy and cell survival was analyzed using flow cytometry together with trypan blue staining. Results In Boyden chamber, the amount of SW480 moving into the second floor was increased under the hypoxic microenvironment. In experimental groups that were exposed to hypoxic conditions, the MDC fluorescence intensity of SW480 cells rndecreased from 20. 53 ± 0. 64 to 17. 00 ± 0. 20, while that of CD133 + CSCs increased from 19. 89 ± 0. 58 to 33. 13 ± 1. 95 ( P <0. 05) . The trypan blue staining showed that under hypoxic conditions, the viability of SW480 cells decreased significantly over time (P <0. 05 ) , while that of CD133 + cells had no significant changes. Conclusions For SW480 cells, the invasion may be enhanced under the hypoxic microenvironment, with both the level of autophagy and viability decreased. For CD133 + cells, the occurrence of autophagy increased under the hypoxic environment, with the viability basically unchanged.
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