摘要
目的研究尿酸对人脐静脉内皮细胞(HUVECs)C反应蛋白(CRP)基因和蛋白表达的影响,并初步探讨其氧化应激机制。方法将不同浓度的尿酸(0、4、8、12、16mg/dL)分别与HUVECs共同孵育12h,以及将12mg/dL尿酸与HUVECs分别作用不同时间(0、6、12、24、48h)后,采用Real-time PCR检测HUVECs CRP mRNA表达;Western blot检测HUVECs CRP蛋白表达;以2′,7′-二氯荧光素二乙酸酯(DCF-DA)为荧光探针,用荧光显微镜和流式细胞术检测HUVECs活性氧(ROS)水平,并观察抗氧化剂N-乙酰半胱氨酸(NAC,10mmol/L)对HUVECs ROS水平、CRP mRNA和蛋白表达的影响。结果 8~16mg/dL尿酸诱导HUVECs CRP mRNA和蛋白表达上调;12mg/dL尿酸与HUVECs作用6h,HUVECs CRP mRNA和蛋白表达明显增加(P<0.05),随着时间延长,CRP mRNA和蛋白表达量逐渐增加,至12h达最高峰(P<0.01)。NAC可显著抑制尿酸对HUVECs ROS水平、CRP mRNA和蛋白表达的影响,使CRP mRNA和蛋白表达接近于正常水平。结论尿酸诱导HUVECs CRP表达,其机制可能与尿酸增加HUVECs ROS水平有关,提示尿酸介导的氧化应激和炎症反应可能与内皮细胞的损伤有关。
Objective To explore the oxidative mechanism of uric acid(UA)induced CRP expression in human umbilical vein endothelial cells.Methods Different concentrations of UA(0mg/dL,4mg/dL,8mg/dL,12mg/dL,16mg/dL)were incubated 12h with HUVECs,and HUVECs were stimulated with 12mg/dL UA for different times(6h,12h,24h,48h).CRP mRNA and protein expression were determined by real-time quantitative polymerase chain reaction(RT-qPCR)and Western blot,respectively;the effects of uric acid on the intracellular reactive oxygen species(ROS)production in HUVECs were measured by fluorescence microscope and flow cytometric analysis using a 2′,7′-Dichlorofluorescin diacetate(DCF-DA)fluorescence probe.The effects of Nacetyl cysteine(NAC)on UA-induced levels of ROS,mRNA and protein of CRP in HUVECs were also observed.Results The results demonstrated that UA could significantly increase the mRNA and protein expression of CRP in HUVECs in time-and concentration-dependent manners.HUVECs were stimulated with 12 mg/dL UA at 6h,mRNA and protein levels of CRP significantly higher than that of control level(P<0.05),reached a peak at 12h(P<0.01).NAC reduced UA-induced levels of ROS,mRNA and protein of CRP in HUVECs compared with those of 12mg/dL UA induced group(P<0.05).Conclusion Uric acid significantly increased mRNA and protein expression of CRP in HUVECs in time-and concentration-dependent manners.Its mechanism may be associated with uric acid induced increasing of ROS levels in endothelial cells,which suggested that the uric acid mediated oxidative stress and inflammation may be involved in the injury of endothelial cells.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2013年第5期717-721,726,共6页
Journal of Sichuan University(Medical Sciences)
关键词
C反应蛋白
活性氧
尿酸
人脐静脉内皮细胞
C-reactive protein
Reactive oxygen species
Uric acid
Human umbilical vein endothelial cells
