去外泌体血清对脂肪干细胞及胃癌细胞生物学活性的影响

Effect of exosome-free serum on biological activity of stem cells and gastric cancer cells

目的研究一种去除血清外泌体(exosomes,Exos)的超离方法,以及去外泌体血清对脂肪干细胞(adipose-derived stem cells,ADSCs)和肿瘤细胞(胃癌细胞)的增殖、衰老、周期和凋亡的影响,为用于外泌体机制研究的细胞培养方法提供新方案。方法超离法去除胎牛血清中的外泌体,Western印迹法检测外泌体的去除效果,使用去外泌体血清作为实验组以及正常血清作为对照组配置培养基,对培养的脂肪干细胞和肿瘤细胞用CCK-8法分析细胞生长情况,β-Gal半乳糖苷酶染色法分析细胞衰老情况,流式细胞分析法检测细胞周期及细胞凋亡情况。结果Western印迹法结果显示超离法能高效去除血清中外泌体,对培养的细胞检测分析显示实验组ADSCs增殖减慢,细胞周期,衰老和凋亡均无明显变化。实验组肿瘤细胞的增殖、衰老、细胞周期、凋亡均不存在显著性差异。结论超离法能有效去除血清外泌体对ADSCs和肿瘤细胞外泌体研究的干扰,且对细胞不产生影响。

Objective To investigate the effect of exosome-free serum on the proliferation,senescence,cell cycle and apoptosis of adipose-derived stem cells(ADSCs)and tumor cells.Methods The exosomes in fetal bovine serum(FBS)were removed by ultrafiltration,and the expression of exosome proteins were detected by Western blotting.Exosome-free serum was used for culture of ADSCs and gastric cancer MGC803 cells as study group and normal serum was used as the control group.Cell viability was measured by CCK-8 assay,aging cells were identified byβ-Gal galactosidase staining,cell cycle and apoptosis were detected by flow cytometry.Results Western blotting showed that the ultrafiltration was able to effectively remove serum exosomes.The proliferation of ADSCs in the study group was slowed down,while there is no significant change in senescence,cell cycle and apoptosis.There were no significant differences in proliferation,senescence,cell cycle and apoptosis of tumor cells in the study group.Conclusion Ultrafiltration can effectively remove serum exosomes which can prevent the interference in the study of the exosomes and has no effects on the cell quality.