不同浓度尿酸盐对人肝THLE-2细胞钙代谢的影响及机制研究

Effects of different concentrations of urate on calcium metabolism in human liver THLE-2 cells

目的:观察不同浓度尿酸盐对人肝THLE-2细胞钙代谢的影响及机制。方法:体外培养人肝THLE-2细胞,取生长状态佳且处于对数生长期的THLE-2细胞,将其分为A组(阴性对照组)、B组(360 mol/L尿酸盐)、C组(600 mol/L尿酸盐)三个实验组;各组干预THLE-2细胞48 h后,采用Fluo-3/AM荧光探针测定细胞内Ca^(2+)浓度,实时定量RT-PCR及Western blot法检测细胞三磷酸肌醇受体(IP3R)mRNA及蛋白表达。结果:B组与C组中THLE-2细胞内Ca^(2+)浓度均高于A组(均P<0.05),B组与C组中IP3R mRNA的表达水平均高于A组(均P<0.05),B组与C组中THLE-2细胞中IP3R蛋白表达水平均高于A组(均P<0.05);B组IP3R mRNA和蛋白表达水平均低于C组(均P<0.05)。结论:高尿酸血症可明显影响人肝THLE-2细胞钙离子水平,其机制可能与升高THLE-2细胞IP3R mRNA及蛋白表达水平、影响内质网及线粒体钙稳态有关。

Objective:To observe the effect of different concentrations of urate on calcium metabolism of hepatocytes and its mechanism.Methods:Liver THLE-2 cells were cultured in vitro,and THLE-2 cells with good growth condition and logarithmic growth stage were selected and divided into group A(negative control group),group B(360 mol/L urate)and group C(600 mol/L urate).After 48 hours intervention in THLE-2 cells,the intracellular Ca 2+concentration was determined by Fluo-3/AM fluorescence probe,and the mRNA and protein expression of inositol triphosphate receptor(IP3R)were detected by real-time quantitative RT-PCR and Western blot.Results:The concentration of Ca 2+in THLE-2 cells in group B and C was higher than that in the control group(all P<0.05).The expression level of IP3R mRNA in group B and C was higher than that in group A(all P<0.05).IP3R protein concentration in THLE-2 cells in group B and C was higher than that in group A(all P<0.05).The expression levels of IP3R mRNA and protein in group B were lower than those in group C(all P<0.05)Conclusion:Hyperuricemia can significantly affect the calcium ion level in liver THLE-2 cells,and the mechanism may be related to the increase of IP3R mRNA and protein expression levels in liver THLE-2 cells,and the influence of calcium homeostasis in endoplasmic reticulum and mitochondria.