不同浓度尿酸对体外培养人脐静脉血管内皮细胞增殖过程的影响

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。

Effects of uric acid in different concentrations on the proliferation of human umbilical vein endothelial cells in vitro

BACKGROUND:Recently, there is more and more attention on uric acid (UA), especially the correlation with cardiovascular disease in the filed of epidemiology; however, researches of the effect of UA on endothelial cells are lack based on cytology.OBJECTIVE:To explore the effects of UA in different concentrations on human umbilical vein endothelial cells line (ECV304), malondialdehyde (MDA), nitric oxide (NO) and 3-(4,5-dimethyl thiazole-2)-2, 5-diphenyl thiazolyl blue (MTT)in vitro.DESIGN: Randomized controlled study based on ECV304.5ETTING: Department of Endocrine, Xijing Hospital, the Fourth Military Medical University of Chinese PLA.MATERIALS:ECV304 was provided by Department of Immunology, the Fourth Military Medical University of Chinese PLA; DMEM Iow-glucose medium by Gibco Company, USA; bovine serum by Beijing Yuanheng Shengma Biotechnology Researching Institute; trypsin by Gibco Company, USA; MTT by Huamei Company; dimethyl sulfoxide (DMSO) analytical pure by Tianjin Bodi Chemical Engineering Co. Ltd.; UA by Sigma Company; NO kit and MDA kit by Nanjing Jiancheng Company; ordinary invert microscope and enzyme-linked immune detector IX70 invert microscope by Olympus, Japan;enzyme-linked immune detector by Eastern China Electron Tube Factory.METHODS: The experiment was carried out in Department of Endocrine and Burning, the Fourth Military Medical University of Chinese PLA from December 2003 to April 2004. Resuscitation, culture, regeneration and inoculation of endothelia cells were undertaken Cell Culture published by Situ et al. Endothelia cells were cultured with non-serum DMEM for 24hours so as to maintain synchronization at the phase of G0/G1 and divided into 4 groups, including control group, low-concentration UA group, moderate-concentration UA group and high-concentration UA group. Each group was divided into 3time points, including 24 hours, 48 hours and 72 hours with 8 samples in each time point. Samples were added with 5％serum medium containing 0 mmol/L, 0.1 mmol/L, 0.2 mmol/L and 0.4 mmol/L UA in control group, low-concentration UA group, moderate-concentration UA group and high-concentration UA group, respectively, and incubated in box with the volume fraction of 0.05 CO2 at 37 ℃. Twenty-four hours later, MDA and MTT were detected; additionally, MTT was detected once more after 48 and 72 hours.MAIN OUTCOME MEASURES: Proliferation of ECV304 and content of NO and MDA.RESULTS:With the increasing concentration of UA, content of MDA was decreased to (2.97±0.05),(2.89±0.09),(2.78±0.10) and (2.44±0.03) μmol/L, respectively; content of NO was (6.86±1.41), (12.5±2.7), (18.9±1.8) and (21.1±1.4) μ mol/L. Absorbencies of NO and MTT were increased and proliferation was increased remarkably at 48 hour.CONCLUSION: A which is characterized by anti-oxidation may promote proliferation of vascular endothelia cells and release of NO.