凝血酶对骨髓来源内皮祖细胞增殖能力的影响

Effect of thrombin on proliferation of bone-marrow derived endothelial progenitor cells

目的:观察凝血酶对骨髓来源内皮祖细胞(EPCs)增殖能力的影响并探讨其机制。方法:密度梯度离心法从大鼠骨髓分离EPCs,培养7 d后收集贴壁细胞并加入不同浓度凝血酶(10-2,10-1,1,10,100 U/mL)干预一定时间(6,12,24,48 h)。CCK-8试剂盒检测EPCs增殖。实时定量PCR检测凝血酶刺激以及NF-κB抑制剂预处理以后EPCs的VEGF mRNA表达变化,ELISA法检测其分泌量变化。结果:凝血酶干预组的细胞增殖数均高于对照组,10 U/mL凝血酶作用24 h对内皮祖细胞数量影响最为显著(P<0.05)。与对照组相比,凝血酶刺激后VEGFmRNA表达及分泌量均增高,使用NF-κB抑制剂预处理后可以抑制凝血酶的这种作用。结论:凝血酶可以通过NF-κKB途径上调VEGF的表达,促进EPCs增殖。

Objective： To investigate the effects of thrombin on proliferation of bone-marrow de- rived endothelial progenitor cells （EPCs）and the underlying mechanism. Methods： EPCs were acquired from the bone marrow of SD rats by density gradient centrifugation. After 7 days of culture, EPCs were incubated with thrombin in different concentrations（10-2,10 -1, 1,10,100 U/mL）for different time points （6,12,24,48 h）. EPCs proliferation was measured by Cell Counting Kit-8 Real time PCR was used to measure VEGF mRNA expression of EPCs after intervention by thrombin or pretreatment with NFkB inhibitor. ELISA assay was used to measure VEGF secretion. Results： EPCs proliferation was significant higher in the thrombin intervention group compared with control group, it reached a maximum at 10 U/mL after 24h of intervention （P〈0.05）. Thrombin up-regulated VEGF mRNA level and increased the se cretion, which process can be blocked by NFkB inhibitor. Conclusion： Thrombin could induce the proliferation of EPCs via increasing the expression of VEGF through NFkB pathway.