Effect of Intestinal Flora Metabolites Deoxycholic Acid on the Proliferation and Cell Cycle of Human Umbilical Cord Mesenchymal Stem Cells
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摘要:
目的 探讨肠道菌群代谢物次级胆汁酸DCA不同浓度和不同作用时间在体外环境下对人脐带间充质干细胞（human umbilical cord mesenchymal stem cells，hUC-MSCs）增殖和细胞周期的影响。 方法 CCK8及流式细胞仪检测在含不同浓度DCA（0.00 μmol/L、1.56 μmol/L、3.13 μmol/L、6.25 μmol/L、12.50 μmol/L、25.00 μmol/L、50.00 μmol/L、100.00 μmol/L、200.00 μmol/L、400.00 μmol/L、800.00 μmol/L）干细胞培养基中培养hUC-MSCs 24 h、48 h、72 h后，干细胞增殖及细胞周期的变化。 结果 脱氧胆酸对hUC-MSCs增殖的影响体现在DCA剂量和作用时间两方面（两者交互作用，F = 6.622，P < 0.001）；当浓度一定时，时间越长，细胞增殖越多；当作用时间一定时，浓度越高，细胞增殖抑制。浓度一定时，随时间的增加，细胞阻滞在G0/G1期的比例增加（P < 0.001）；不同作用时间DCA对 hUC-MSCs的细胞周期表现为低浓度组细胞阻滞在G0/G1期和S期，高浓度组细胞阻滞在S期和G2/M期（P < 0.001）。 结论 体外环境下，DCA对hUC-MSCs增殖及细胞周期的影响与浓度和作用时间相关。 Abstract:Objective To investigate the influence of intestinal flora metabolites secondary bile acid DCA at different concentration and reaction time on the proliferation and cell cycle of human umbilical cord mesenchymal stem cells （hUC-MSCs） in vitro. Methods CCK8 and flow cytometry were used to detect the changes in cell proliferation and cell cycle of hUC-MSCs cultured in stem cell medium containing different concentrations of DCA （0.00 μmol/L, 1.56 μmol/L, 3.13 μmol/L, 6.25 μmol/L, 12.50 μmol/L, 25.00 μmol/L, 50.00 μmol/L, 100.00 μmol/L, 200.00 μmol/L, 400.00 μmol/L, 800.00 μmol/L） at 24h, 48h and 72h. Results The effect of deoxycholic acid on the proliferation of hUC-MSCs was both dose- and time-dependent （interaction, F = 6.622, P < 0.001）．When the concentration was constant, cell proliferation increases with time; When the treatment time was fixed, the cell proliferation decreased with the increase of concentration. When the concentration was constant, the proportion of cell was arrested in G0/G1 phase increased with time （P < 0.001）．The cell cycle of hUC-MSCs treated by DCA was blocked in G0/G1 and S phase in low concentration group, while S and G2/M phase in high concentration group （P < 0.001）． Conclusion The effects of DCA on the proliferation and cell cycle of hUC-MSCs were both dose and time in vitro. -
图 1 脱氧胆酸不同浓度及不同作用时间对 hUC-MSCs 增殖的影响
A：脱氧胆酸不同浓度及不同作用时间对 hUC-MSCs 增殖的影响；B：不同浓度脱氧胆酸作用 24 h 时 hUC-MSCs增殖情况；C：不同浓度脱氧胆酸作用48 h时hUC-MSCs增殖情况；D：不同浓度脱氧胆酸作用72 h时hUC-MSCs 增殖情况。各浓度组与0.00 μmol/L浓度组比较，ns表示差异无统计学意义，*表示 P < 0.05，**表示 P < 0.01，***表示 P < 0.001，****表示 P < 0.0001。
Figure 1. Effects of DCA with different concentration and different time on the proliferation of hUC- MSCs
图 2 脱氧胆酸不同浓度及不同作用时间 hUC-MSCs 细胞周期分布情况
Figure 2. Cell cycle proportion of hUC-MSCs with different concentration and different time of DCA
图 3 不同浓度及不同作用时间脱氧胆酸刺激后流式细胞技术检测 hUC-MSCs 细胞周期代表图例
A~D：24 h时脱氧胆酸0.00 μmol/L～400.00 μmol/L hUC-MSCs细胞周期分布情况；E~H：48 h时脱氧胆酸0.00 μmol/L～400.00 μmol/L hUC-MSCs细胞周期分布情况；I~L：72 h时脱氧胆酸0.00 μmol/L～400.00 μmol/L hUC-MSCs细胞周期分布情况。
Figure 3. Cell cycle of hUC-MSCs tested by flow cytometry after different concentration and different time of DCA
表 1 脱氧胆酸不同浓度及不同作用时间hUC-MSCs吸光度值（OD450nm值）（
$\bar x \pm s $ ）Table 1. Absorbance （OD450nm） of hUC-MSCs at different dose and time of DCA （
$\bar x \pm s $ ）脱氧胆酸浓度（μmol/L） 样本数 24 h 48 h 72 h 0.00（对照组） 45 0.61 ± 0.12 0.77 ± 0.14* 0.97 ± 0.32○* 1.56 45 0.51 ± 0.07 0.79 ± 0.19* 1.23 ± 0.24○* 3.13 45 0.54 ± 0.08 0.79 ± 0.21* 1.01 ± 0.31○* 6.25 45 0.56 ± 0.09 0.80 ± 0.16* 1.05 ± 0.33○* 12.50 45 0.55 ± 0.05 0.81 ± 0.07* 1.18 ± 0.28○* 25.00 45 0.55 ± 0.07 0.78 ± 0.13* 1.21 ± 0.20○* 50.00 45 0.57 ± 0.08 0.77 ± 0.03* 1.07 ± 0.24○* 100.00 45 0.58 ± 0.08 0.75 ± 0.16* 1.05 ± 0.23○* 200.00 44 0.58 ± 0.09 0.72 ± 0.16 0.84 ± 0.26* 400.00 45 0.50 ± 0.10 0.54 ± 0.19 0.53 ± 0.17 800.00 45 0.49 ± 0.12 0.48 ± 0.18 0.49 ± 0.18 与24 h比较，*P < 0.05；与48 h比较，○P < 0.05。 
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表 2 脱氧胆酸不同浓度及不同作用时间 hUC-MSCs 细胞周期比例（%）
Table 2. Cell cycle ratio of hUC-MSCs with different concentration and different time of DCA （%）
时间
（h）分期 0.00
μmol/L1.56
μmol/L25.00
μmol/L400.00
μmol/L24 G0/G1期 77.60 82.33* 83.77* 48.17* S期 16.48 17.67 16.23 51.83* G2/M期 5.91 0.00* 0.00* 0.00* 48 G0/G1期 87.07△ 92.85*△ 85.73*△ 42.99*△ S期 5.45△ 2.92*△ 14.27*△ 50.89* G2/M期 7.48△ 4.23*△ 0.00* 6.12*△ 72 G0/G1期 97.60△ 98.19*△ 98.41*△ 73.14*△ S期 2.40△ 1.81*△ 1.59*△ 22.70*△ G2/M期 0.00△ 0.00 0.00 4.16*△ 与0.00 µmol/L比较，*P < 0.0001；与24 h比较，△P < 0.0001。
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