鞘氨醇激酶1/1-磷酸鞘氨醇信号对老年脐静脉内皮细胞增殖及迁移能力的影响研究

Effect of SPK1 and S1P on proliferation and migration of old human umbilical vein endothelial cells

目的:探讨鞘氨醇激酶1(sphingosine kinase 1,SPK1)及1-磷酸鞘氨醇(sphingosine-1-phosphate,S1P)对老年脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖及迁移能力的影响。方法:通过实验确定pLKO.1-shSPK1-GFP干涉慢病毒及pPIG-U6-Scramble-GFP对照慢病毒的最佳感染复数(multiplicity of infection,MOI),并用最佳MOI感染HUVECs。48 h后提取细胞的蛋白质及RNA,蛋白免疫印迹法(western blot,WB)及实时荧光定量PCR(real-time PCR,RT-PCR)检测HUVECs内SPK1的蛋白和基因表达水平。用SPK1干涉慢病毒及对照病毒或S1P处理HUVECs,细胞活性检测试剂盒(cell counting kit-8,CCKit-8)检测细胞的增殖能力,Transwell检测细胞的迁移能力。结果:用最佳感染复数20 MOI的慢病毒感染HUVECs 48 h后,其SPK1的蛋白和基因表达水平均显著下调50%左右(P<0.01或P<0.05)。CCKit-8增殖实验及Transwell迁移实验表明,干涉SPK1可以显著抑制HUVECs的增殖及迁移能力(P<0.01)。而100 nM及200 nM S1P则可以显著促进HUVECs的增殖及迁移能力(P<0.01)。结论:HUVECs内SPK1表达水平的下降可以显著抑制HUVECs的增殖及迁移能力,而其催化产物S1P则可以促进HUVECs的增殖及迁移能力。

Objective:To investigate the effect of sphingosine kinase 1(SPK1) and sphingosine-1-phosphate(S1 P) on the proliferation and migration of old human umbilical vein endothelial cells(HUVECs).Methods:To determine the optimal multiplicity of infection(MOI) of pLKO.1-shSPK1-GFP interfering lentivirus and pPIG-U6-Scramble-GFPlentivirus and infected HUVECs with them.After 48 h,collecting protein and RNA of HUVECs,and detect the protein and gene expression of SPK1 in HUVECs by western blotting(WB) and real-time PCR(RT-PCR).After that,treating HUVECs with pLKO.1-shSPK1-GFP interfering lentivirus,pPIG-U6-Scramble-GFP lentivirus or S1 P,the effect of interfering SPK1 and S1 P on proliferation and migration were examined via cell counting kit-8(CCKit-8) assay and transwell.Results:The protein and gene expression of SPK1 were significantly decreased about 50%(P<0.01 or P<0.05) after infected with pLKO.1-shSPK1-GFP interfering lentivirus about 48 h.CCKit-8 and transwell assay showed that interfering SPK1 could significantly inhibit the proliferation and migration of HUVECs(P<0.01).What′s more,100 nM and 200 nM S1 P could significantly promote the proliferation and migration of HUVECs(P<0.01).Conclusion:The reducing expression of SPK1 in HUVECs can significantly inhibit the proliferation and migration of HUVECs,and its catalysate S1 P can promote them.