活细胞硫化氢检测新方法

发布时间：2018-04-04 20:01

  本文选题：硫化氢　切入点：细胞　出处：《北京大学学报(医学版)》2013年03期

【摘要】：目的:建立测定活细胞释放微量硫化氢(hydrogen sulfide,H2S)的简易方法。方法:在细胞培养板盖上方黏附滤膜,细胞释放的硫化氢与滤膜上的醋酸锌反应产生硫化锌,采用亚甲基蓝分光光度法测定并计算细胞释放的硫化氢的量。应用该方法分别检测了HepG2细胞和人脐静脉内皮细胞硫化氢的释放量。结果:HepG2细胞添加L-半胱氨酸以及辅酶磷酸吡哆醛后,继续培养12 h,H2S释放量为(859.39±19.12)nmol/(min.106cells);给予胱硫醚-γ-裂解酶抑制剂炔丙基甘氨酸(DL-propargylglycine,PAG)后,H2S产率下降到(341.34±105.90)nmol/(min.106cells);胱硫醚-β-合酶抑制剂间羟胺处理后,H2S产率下降到(375.05±174.50)nmol/(min.106cells);两种酶抑制剂联合使用后H2S释放量显著抑制,为(204.47±97.14)nmol/(min.106cells)。人脐静脉内皮细胞也可内源性产生H2S,HUVEC细胞H2S的释放量为(26.23±3.24)nmol/(min.106cells),约为HepG2细胞产量的1/30。台盼蓝检测细胞活性大于95%,细胞生长状态良好,表明该方法无细胞毒作用,细胞可根据实验需要继续培养。结论:滤膜吸附法简便易行、实用可靠,可应用于活细胞释放硫化氢的测定。
[Abstract]:Objective: to establish a simple method for the determination of trace hydrogen sulfide (H _ 2S) released from living cells.Methods: the membrane was adhered to above the cell culture plate. The hydrogen sulfide released by the cell reacted with zinc acetate on the filter membrane to produce zinc sulfide. The amount of hydrogen sulfide released by the cell was determined and calculated by methylene blue spectrophotometry.The release of hydrogen sulfide from HepG2 cells and human umbilical vein endothelial cells (HUVEC) was measured by this method.Results after addition of L- cysteine and coenzyme pyridoxal,缁х画鍩瑰吇12 h,H2S閲婃斁閲忎负(859.39卤19.12)nmol/(min.106cells);缁欎簣鑳辩～閱，

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