Biologic effects of different concentrations of putrescine on human umbilical vein endothelial cells
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摘要: 目的 探讨不同浓度腐胺对人脐静脉内皮细胞(HUVEC)增殖、迁移、凋亡的影响。 方法 常规培养HUVEC,取第3~5代细胞进行以下实验。(1)按随机数字表法(分组方法下同)将细胞分为500、1 000、5 000 μg/mL腐胺组,每组3孔,分别以含相应浓度腐胺的完全培养液培养24 h后,采用倒置光学显微镜观察细胞形态。(2)将细胞分为0.5、1.0、5.0、10.0、50.0、100.0、500.0、1 000.0 μg/mL腐胺组及对照组,每组4孔。各腐胺组细胞采用含相应浓度腐胺的完全培养液、对照组采用不含腐胺的完全培养液培养24 h后,采用比色法测定细胞增殖活性,结果以吸光度值表示。(3)同实验(2)将细胞分组(每组1孔)及培养后,Transwell迁移实验测定细胞迁移能力。(4)同实验(2)将细胞分组(每组1瓶)及培养后,采用流式细胞仪测定细胞凋亡率。对数据进行单因素方差分析、Kruskal-Wallis检验、Dunnett检验。 结果 (1)培养24 h后,500 μg/mL腐胺组细胞贴壁良好,形态无明显改变;1 000 μg/mL腐胺组细胞贴壁减少,细胞变小、变圆;5 000 μg/mL腐胺组细胞未贴壁、呈碎片状。(2)0.5、1.0、5.0、10.0、50.0、100.0、500.0、1 000.0 μg/mL腐胺组及对照组细胞增殖活性分别为0.588±0.055、0.857±0.031、0.707±0.031、0.662±0.023、0.450±0.019、0.415±0.014、0.359±0.020、0.204±0.030、0.447±0.021,组间总体比较差异明显(
χ 2=6.86,P =0.009)。0.5、1.0、5.0、10.0 μg/mL腐胺组细胞增殖活性较对照组升高(P <0.05或P <0.01),500.0、1 000.0 μg/mL腐胺组细胞增殖活性较对照组降低(P 值均小于0.01),50.0、100.0 μg/mL腐胺组细胞增殖活性与对照组相近(P 值均大于0.05)。(3)各腐胺组及对照组迁移细胞数组间总体比较差异明显(F =138.662,P <0.001)。1.0、5.0、10.0 μg/mL腐胺组迁移细胞数多于对照组(P 值均小于0.01),500.0、1 000.0 μg/mL腐胺组迁移细胞数少于对照组(P 值均小于0.01),0.5、50.0、100.0 μg/mL腐胺组迁移细胞数与对照组相近(P 值均大于0.05)。(4)各腐胺组及对照组细胞凋亡率组间总体比较差异明显(χ 2=3.971,P =0.046)。0.5、1.0、5.0、10.0 μg/mL腐胺组细胞凋亡率较对照组降低(P 值均小于0.05),500.0、1 000.0 μg/mL腐胺组细胞凋亡率较对照组升高(P 值均小于0.01),50.0、100.0 μg/mL腐胺组细胞凋亡率与对照组相近(P 值均大于0.05)。 结论 低微浓度的腐胺能显著促进HUVEC增殖与迁移;而较高浓度的腐胺能显著抑制HUVEC增殖与迁移,并促进细胞凋亡。Abstract: Objective To explore the effects of different concentrations of putrescine on proliferation, migration, and apoptosis of human umbilical vein endothelial cells (HUVECs). Methods HUVECs were routinely cultured in vitro. The 3rd to the 5th passage of HUVECs were used in the following experiments. (1) Cells were divided into 500, 1 000, and 5 000 μg/mL putrescine groups according to the random number table (the same grouping method was used for following grouping), with 3 wells in each group, which were respectively cultured with complete culture solution containing putrescine in the corresponding concentration for 24 h. Morphology of cells was observed by inverted optical microscope. (2) Cells were divided into 0.5, 1.0, 5.0, 10.0, 50.0, 100.0, 500.0, 1 000.0 μg/mL putrescine groups, and control group, with 4 wells in each group. Cells in the putrescine groups were respectively cultured with complete culture solution containing putrescine in the corresponding concentration for 24 h, and cells in control group were cultured with complete culture solution with no additional putrescine for 24 h. Cell proliferation activity (denoted as absorption value) was measured by colorimetry. (3) Cells were divided (with one well in each group) and cultured as in experiment (2), and the migration ability was detected by transwell migration assay. (4) Cells were divided (with one flask in each group) and cultured as in experiment (2), and the cell apoptosis rate was determined by flow cytometer. Data were processed with one-way analysis of variance, Kruskal-Wallis test, and Dunnett test. Results (1) After 24-h culture, cell attachment was good in 500 μg/mL putrescine group, and no obvious change in the shape was observed; cell attachment was less in 1 000 μg/mL putrescine group and the cells were small and rounded; cells in 5 000 μg/mL putrescine group were in fragmentation without attachment. (2) The absorption values of cells in 0.5, 1.0, 5.0, 10.0, 50.0, 100.0, 500.0, 1 000.0 μg/mL putrescine groups, and control group were respectively 0.588±0.055, 0.857±0.031, 0.707±0.031, 0.662±0.023, 0.450±0.019, 0.415±0.014, 0.359±0.020, 0.204±0.030, and 0.447±0.021, with statistically significant differences among them (χ 2=6.86,P =0.009). The cell proliferation activity in 0.5, 1.0, 5.0, and 10.0 μg/mL putrescine groups was higher than that in control group (P <0.05 orP <0.01). The cell proliferation activity in 500.0 and 1 000.0 μg/mL putrescine groups was lower than that in control group (withP values below 0.01). The cell proliferation activity in 50.0 and 100.0 μg/mL putrescine groups was close to that in control group (withP values above 0.05). (3) There were statistically significant differences in the numbers of migrated cells between the putrescine groups and control group (F =138.662,P <0.001). The number of migrated cells was more in 1.0, 5.0, and 10.0 μg/mL putrescine groups than in control group (withP value below 0.01). The number of migrated cells was less in 500.0 and 1 000.0 μg/mL putrescine groups than in control group (withP value below 0.01). The number of migrated cells in 0.5, 50.0, and 100.0 μg/mL putrescine groups was close to that in control group (withP values above 0.05). (4) There were statistically significant differences in the apoptosis rate between the putrescine groups and control group (χ 2=3.971,P =0.046). The cell apoptosis rate was lower in 0.5, 1.0, 5.0, and 10.0 μg/mL putrescine groups than in control group (withP values below 0.05). The cell apoptosis rate was higher in 500.0 and 1 000.0 μg/mL putrescine groups than in control group (withP values below 0.01). The cell apoptosis rates in 50.0 and 100.0 μg/mL putrescine groups were close to the cell apoptosis rate in control group (withP values above 0.05). Conclusions Low concentration of putrescine can remarkably enhance the ability of proliferation and migration of HUVECs, while a high concentration of putrescine can obviously inhibit HUVECs proliferation and migration, and it induces apoptosis.-
Key words:
- Putrescine /
- Endothelial cells /
- Cell proliferation /
- Cell migration assays /
- Apoptosis
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