C反应蛋白对人外周血来源内皮祖细胞Notch信号通路表达影响的实验研究

目的观察C反应蛋白(cRP)对内皮祖细胞(EPC)中Notch信号通路表达的影响。方法密度梯度离心法获取人外周血单个核细胞并培养,FITC-荆豆凝集素I、DiI-乙酰化低密度脂蛋白荧光双染进行鉴定。取生长7～14 d EPC,分为对照组、10和20 mg/L CRP干预组,培养48 h后分别采用四氮唑溴盐比色法、改良的Boyden小室和黏附能力检测各组EPC增殖、迁移和粘附能力。采用RT-PCR检测不同浓度CRP对EPC中Notch信号通路mRNA的影响,Western blot检测各组Notch-1及其配体Jagged-1的蛋白基因表达。结果外周血分离获取的单个核细胞经体外培养后形成了典型的EPC集落,经荧光双染证实为EPC。CRP(10、20 mg/L)组EPC数量分别为(61±3)、(54±3)个,对照组EPC数量为(71±4)个。CRP各组EPC在490 nm吸光度值分别为0.287±0.046、0.211±0.042,对照组为0.386±0.044。与对照组相比,CRP各组EPC黏附数量和迁移数量均显著减少(P<0.05)。CRP处理48 h后EPC中Jagged-1和Notch-1 mRNA含量明显增加,其中10和20 mg/L组Jagged-1 mRNA分别升高了3.84和10.70倍,Notch-1 mRNA分别升高了2.97和3.58倍;差异均具有统计学意义(P<0.05)。10和20 mg/LCRP处理组Jagged-1和Notch-1蛋白表达均高于对照组(P<0.05)。结论 CRP处理后EPC的Notch信号通路表达发生了显著的改变,提示该通路可能是CRP影响EPC生物学功能的机制之一。

Effect of C-reactive protein on Notch pathway components in human periphery blood endothelial progenitor cells

Objective To observe the effect of C-reactive protein(CRP) on the expressions of Notch pathway components in human peripheral blood endothelial progenitor cells(EPC) in vitro.Methods Mononuclear cells isolated by density gradient centrifugation of human peripheral blood mixed with 6%hydroxyethyl starch(Hes) were plated on fibronectin-coated 6-well culture dishes.After 7 days,the adherent cells were cultured in the presence of 10 and 20 mg/L CRP for 48 h,and the proliferation,migration,and adhesion abilities of the cells were observed.The mRNA expressions of Notch-1 and its ligand Jagged-1 in the EPCs were measured by RT-PCR,and their protein expressions by Western blotting.Results CRP at 10 and 20 mg/L caused a significant reduction in the number of viable EPCs(61±3 and 54±3,respectively) as compared with PBS(71±4,P< 0.05).CRP also resulted in a significant suppression of the proliferation,migration and adhesion capacities of the EPCs.The mRNA and protein expressions of Jagged-1 and Notch-1 in the EPCs significantly increased following CRP exposure in comparison with PBS treatment.Conclusion CRP can suppress the proliferation,migration and adhesion capacities of the EPCs probably by affecting the expressions of the Notch-1 pathway components.