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目的 :建立抵抗失巢凋亡的结肠癌细胞系,并分析其细胞生物学特性。方法 :通过贴壁-悬浮交替培养的方法筛选出抗失巢凋亡的结肠癌SW480细胞(命名为SW480-R),采用FCM法鉴定失巢条件下细胞凋亡的改变,然后应用MTT法、细胞划痕愈合实验和Transwell小室法分别检测细胞增殖、迁移和侵袭能力的变化,最后应用蛋白质印迹法检测上皮-间质转化(epithelial-mesenchymal transition,EMT)标志物的表达水平。结果 :经过5次悬浮-贴壁培养后,建立抗失巢凋亡细胞株SW480-R。悬浮培养48和72 h后,SW480-R细胞的凋亡率均明显低于SW480细胞(P<0.05,P<0.01)。贴壁培养12、24和48 h后,SW480-R细胞的增殖活性均低于SW480细胞(P值均<0.05)。SW480-R细胞的迁移和侵袭能力均明显高于SW480细胞(P值均<0.05)。SW480-R细胞中上皮细胞标志物E-cadherin表达下调,间质细胞标志物vimentin表达水平上调(P值均<0.05)。结论 :成功建立了抗失巢凋亡细胞株SW480-R。与亲本细胞SW480相比,SW480-R细胞的增殖速度减慢,而侵袭和迁移能力增强,其机制可能与EMT调控有关。
OBJECTIVE: To establish a colon cancer cell line resistant to anoikis and analyze its biological characteristics. Methods: SW480 cells (named as SW480-R) were screened for anti-anaerobic colon cancer cell line SW480-R by adherent-suspension culture. FCM was used to identify the changes of apoptosis in anaerobic condition. The changes of cell proliferation, migration and invasion were detected by cell scratch healing assay and Transwell chamber assay. Finally, the expression of epithelial-mesenchymal transition (EMT) markers was detected by Western blotting. Results: After five times suspension-adherent culture, an anti-anoikis cell line SW480-R was established. The apoptosis rate of SW480-R cells was significantly lower than that of SW480 cells (P <0.05, P <0.01) at 48 and 72 h after suspension culture. After adherent culture for 12, 24 and 48 h, the proliferation activities of SW480-R cells were lower than those of SW480 cells (all P <0.05). The migration and invasion ability of SW480-R cells were significantly higher than that of SW480 cells (all P <0.05). The expression of E-cadherin was down-regulated in epithelial cells and the expression of vimentin was up-regulated in SW480-R cells (all P <0.05). Conclusion: An anti-anoikis cell line SW480-R was successfully established. Compared with SW480 parental cells, SW480-R cells showed slower proliferation and increased invasion and migration, which may be related to EMT regulation.