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Effects of secreted endostatin from liver stem cell as vector on proliferation and apoptosis of vascular endothelial cells in vitro
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摘要: 目的观察携带内皮抑素基因的载体肝干细胞分泌表达的内皮抑素蛋白（Endostatin,ES）对血管内皮细胞体外增殖和凋亡的影响。方法体外扩增培养本室构建的携带内皮抑素基因的载体肝干细胞WB-ES,收集含有分泌型Endostatin的上清液。将人脐静脉内皮细胞ECV304体外增殖培养,加入不同浓度的含倍比稀释Endostatin的上清液,在不同的作用时间（24、48、72 h）,通过四甲基偶氮唑蓝比色法（MTT）检测细胞生长抑制率。采用流式细胞仪检测细胞周期和凋亡率,分析载体肝干细胞WB-ES分泌表达的Endostatin对ECV304的增殖和凋亡的影响。结果载体肝干细胞胞外表达的Endostatin对人脐静脉内皮细胞ECV304生长有显著抑制作用,48 h作用达到高峰,随浓度增加抑制作用增强;Endostatin作用的实验组,G1期细胞比例增加,S期细胞数下降,抑制细胞生长的机制可能主要是通过对细胞增殖的影响（P<0.05）;实验组细胞存在细胞凋亡增加现象,但与对照组比较凋亡率差异无统计学意义。结论携带内皮抑素基因的载体肝干细胞WB-ES胞外表达的Endostatin在体外可有效抑...Abstract: Objective To observe the influence of secreted endostatin (ES) from liver stem cells as gene therapy vector on proliferation and apoptosis of vascular endothelial cells.Methods To establish liver stem cells by recombinant adenovirus, which could express endostatin stably, and were named WB-ES by our department.WB-ES cells were cultured to acquire the supernatant including endostatin.Then, to add the different concentration supernatant into ECV304 cells for further culturing for 24 h, 48 h, 72 h respectively, and equivalent PBS was added in the control group.The inhibitory effect of supernatant was measured by methyl thiazolyl tetrazolium (MTT) assay.Cell cycle analysis of the experiment group and the control group ECV304 cells was detected by flow cytometry, and early apoptosis was detected by Annexin V-FITC immunofluorescence flow cytometry.Results Free endostatin inhibited the proliferation of ECV304 cells in vitro, which was expressed in the supernatant of liver stem cell WB-ES.Inhibitory effect had the features of concentration dependent and reached the summit at 48 h.In the experiment group, endostatin induced the G1 phase block of cell cycle and decrease of S phase cells proportion (P<0.05) .Early apoptosis rate was higher in the experiment group, but there was no statistical difference in the two groups.Conclusion secreted endostatin expressed by WB-ES could inhibit the proliferation of endothelial cells effectively, which illustrated the potential ability of WB-ES as a vector for anti-angiogenesis of HCC gene therapy.
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Key words:
- stem cells /
- endostatins /
- endothelium /
- vascular /
- cell cycle
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