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本研究探讨血小板衍生膜微粒(platelet-derived membrane microparticles,PMP)对人脐静脉内皮细胞(hu-man umbilical vein endothelial cells,HUVEC)增殖和凋亡的影响。用不同浓度的凝血酶激活血小板释放出PMP,采用流式细胞术检测PMP的释放量,确定制备PMP时凝血酶的最佳应用浓度。以体外培养HUVEC为载体,通过噻唑蓝(MTT)实验和流式细胞术研究不同浓度的PMP对HUVEC增殖和凋亡的影响。结果表明,2、1.5、1.0和0.5U/ml凝血酶激活血小板后PMP的释放率分别为28.7、47.7、50.1和43.9%;HUVEC的增殖与PMP呈剂量依赖关系。在培养液中添加40μg/mlPMP时,HUVEC增殖是空白对照组的1.8±0.3倍;10μl/ml血管内皮细胞生长因子(VEGF)组的HUVEC增殖是空白对照组的1.9±0.5倍,两组之间无统计学差异,(p>0.05);PMP能抑制HUVEC的凋亡,40μg/mlPMP组的细胞凋亡率为(3.9±0.4)%,明显低于空白对照组的细胞凋亡率(9.4±0.5)%(p<0.05)。VEGF(10μl/ml)对HUVEC细胞的凋亡无明显抑制作用,其凋亡率为(8.0±0.8)%。结论:用1U/ml的凝血酶刺激血小板释放的PMP较为均一,且释放量最大,可促进HUVEC细胞的增殖并抑制其凋亡。
This study was designed to investigate the effects of platelet-derived membrane microparticles (PMP) on the proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs). With different concentrations of thrombin to activate platelet release PMP, using flow cytometry to detect the release of PMP to determine the optimum concentration of thrombin when preparing PMP. HUVECs were cultured in vitro. The effects of different concentrations of PMP on the proliferation and apoptosis of HUVEC were studied by MTT assay and flow cytometry. The results showed that 2, 1.5, 1.0 and 0.5 U / ml thrombin activated platelet release rate of PMP were 28.7,47.7,50.1 and 43.9%; HUVEC proliferation and PMP in a dose-dependent manner. The proliferation of HUVEC was 1.8 ± 0.3 times that of the blank control group when 40 μg / ml PMP was added to the culture medium; the proliferation of HUVEC in 10 μl / ml VEGF group was 1.9 ± 0.5 times of the blank control group, (P> 0.05). PMP inhibited the apoptosis of HUVEC. The apoptosis rate of PMP group was (3.9 ± 0.4)%, which was significantly lower than that of the blank control group (9.4 ± 0.5)% (p <0.05). VEGF (10μl / ml) had no obvious inhibitory effect on the apoptosis of HUVEC cells, the apoptotic rate was (8.0 ± 0.8)%. CONCLUSION: PMP released from platelets stimulated by 1U / ml thrombin is more uniform and has the highest release, which can promote the proliferation and inhibit the apoptosis of HUVECs.