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背景:近年对尿酸特别是其与心血管疾病的研究较多,但多集中在流行病学领域,从细胞学角度探讨尿酸对内皮细胞影响的研究并不多见。目的:观察不同浓度的尿酸对体外培养的人脐静脉内皮细胞株(ECV304)丙二醛,一氧化氮及3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐的影响。设计:以人脐静脉内皮细胞为观察对象,随机对照实验。单位:解放军第四军医大学西京医院内分泌科。材料:脐静脉内皮细胞株ECV304由解放军第四军医大学免疫教研室提供;DMEM低糖培养基,美国Gibco公司生产;胎牛血清,北京元亨圣马生物技术研究所生产;胰蛋白酶,美国Gibco公司生产;3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐,华美公司生产;二甲基亚砜(DM-SO),分析纯,天津博迪化工有限公司生产;尿酸,Sigma公司生产。一氧化氮测试盒,丙二醛测试盒,南京建成公司生产。普通倒置显微镜、酶联免疫检测仪IX70型倒置显微镜,日本Olympus生产;酶联免疫检测仪,华东电子管厂生产。方法:实验于2003-12/2004-04在第四军医大学内分泌科和烧伤科进行。内皮细胞的复苏、培养、传代、接种均参考司徒镇强等主编的《细胞培养》中的方法进行。用无血清DMEM培养24h使细胞同步化于G0/G1期,分4组实验,分别为对照组,低浓度尿酸组,中浓度尿酸组和高浓度尿酸组,每组分为24h,48h和72h3个时间点,每个时间点8个样本。对照组,低浓度尿酸组,中浓度尿酸组和高浓度尿酸组分别加入含有0mmol/L,0.1mmol/L,0.2mmol/L,0.4mmol/L尿酸的5%的血清培养液,然后将培养板放入37℃,体积分数为0.05的CO2孵箱中孵育。在24h后检测丙二醛,一氧化氮及3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐,48h及72h分别再检测3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐。主要观察指标:各组人脐静脉内皮细胞增殖情况及一氧化氮和丙二醛的含量。结果:随尿酸浓度增高,ECV304产生丙二醛的含量逐渐下降,分别为(2.97±0.05),(2.89±0.09),(2.78±0.10),(2.44±0.03)μmol/L。ECV304产生一氧化氮的量分别为(6.86±1.41),(12.5±2.7),(18.9±1.8),(21.1±1.4)μmol/L,ECV304产生一氧化氮的量和3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐吸光度逐渐增加,细胞增殖以48h增加最为明显。结论:尿酸有抗氧化性,可促进血管内皮细胞的增殖及一氧化氮的释放。
BACKGROUND: In recent years, there are many studies on uric acid, especially on cardiovascular diseases. However, most of them are concentrated in the field of epidemiology. It is not uncommon to study the effect of uric acid on endothelial cells from the perspective of cytology. OBJECTIVE: To observe the effects of different concentrations of uric acid on malondialdehyde, nitric oxide and 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenylphosphate in cultured human umbilical vein endothelial cell line (ECV304) Effect of tetrazolium bromide. Design: human umbilical vein endothelial cells as the observation object, a randomized controlled trial. Unit: Xijing Hospital, Fourth Military Medical University, Chinese PLA. MATERIALS: Human umbilical vein endothelial cell line ECV304 was provided by Immunization Department of the Fourth Military Medical University of PLA. DMEM low glucose medium was produced by American Gibco Company. Fetal calf serum was produced by Beijing Institute of Biotechnology, Yuanheng. Trypsin was produced by Gibco ; 3- (4,5-dimethylthiazol-2) -2,5-diphenyl tetrazolium bromide, produced by China and the United States; dimethyl sulfoxide (DM-SO) Chemical Co., Ltd. production; uric acid, Sigma company. Nitric oxide test box, malondialdehyde test box, built in Nanjing company. Ordinary inverted microscope, enzyme-linked immunosorbent IX70 inverted microscope, produced by Japan Olympus; enzyme-linked immunosorbent detector, East China tube plant production. Methods: The experiment was performed at Department of Endocrinology and Burns, Fourth Military Medical University from December 2003 to April 2004. Endothelial cell resuscitation, culture, passage, vaccination are referred to Stuart Zhenjiang and other editors of “cell culture” in the method. The cells were cultured in serum-free DMEM for 24 h to synchronize cells in G0 / G1 phase. The cells were divided into 4 groups: control group, low concentration of uric acid group, medium concentration of uric acid group and high concentration of uric acid group, each group was 24h, 48h and 72h3 A time point, each time point 8 samples. The control group, low concentration of uric acid group, medium concentration of uric acid group and high concentration of uric acid group were added with 0mmol / L, 0.1mmol / L, 0.2mmol / L, 0.4mmol / L uric acid in 5% serum culture medium, Plates were incubated at 37 ° C in a volume fraction of 0.05 CO2 incubator. The malondialdehyde, nitric oxide and 3- (4,5-dimethylthiazol-2) -2,5-diphenyl tetrazolium bromide were detected 24h later, and the content of 3- (4 , 5-dimethylthiazol-2) -2,5-diphenyltetrazolium bromide. MAIN OUTCOME MEASURES: Proliferation of human umbilical vein endothelial cells and content of nitric oxide and malondialdehyde in each group. Results: With the increase of uric acid concentration, the content of malondialdehyde in ECV304 decreased gradually (2.97 ± 0.05), (2.89 ± 0.09), (2.78 ± 0.10) and (2.44 ± 0.03) μmol / L, respectively. The amounts of nitric oxide produced by ECV304 were (6.86 ± 1.41), (12.5 ± 2.7), (18.9 ± 1.8) and (21.1 ± 1.4) μmol / - dimethylthiazol - 2) -2,5 - diphenyl tetrazolium bromide salt absorbance increased gradually, the cell proliferation increased most obviously at 48h. Conclusion: Uric acid has antioxidant activity, which can promote the proliferation of vascular endothelial cells and the release of nitric oxide.