活细胞硫化氢检测新方法  

A new methods for determining hydrogen sulfide release in cultured cells

作　　者：谢静[1,2] 曾强[1] 郑扬[2] 廖锋[2] 徐国恒[2] 唐朝枢[2] 耿彬[2] 

机构地区：[1]中国人民解放军总医院国际医学中心,北京100853 [2]北京大学基础医学院生理学与病理生理学系,北京100191

出　　处：《北京大学学报（医学版）》2013年第3期489-492,共4页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(81170235);"十二五"国家科技支撑计划(2012BAI37B04)项目资助~~

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。Objective： To establish a simple method of measuring hydrogen sulfide （H2S） in cultured living cells. Methods ： Filtration membrane was stuck on the lid of cell culture plate. H2 S released from cultured cells was trapped by zinc acetate to generate ZnS deposition. Then the ZnS trapped in the filtra- tion membrane was measured by methylene blue assay and the HzS production from the living cells was counted according to the standard curve. This simple method was used to access the H2S release in HepG2 （high expression CBS and CSE） and HUVEC （low expression CSE） cell lines. Results： H2S generation in cultured HepG2 cells assayed using the present method was （859.39 ± 19. 12 ） nmol/ （min · 10^6 cells）. PAG （CSE inhibitor）, HA （CBS inhibitor） or the two-inhibitor （PAG ＋ HA） treat- ment significantly lowered H2S release, respectively： （ 341.34 ± 105.90 ） nmol/（ min · 10^6 ceils ）, （375.05 ± 174.50） nmol/（min · 10^6cells）, and （204.47 ±97.14） nmol/（min · 10^6cells）. The H2S production of HUVEC was （26.23 ± 3.24） nmol/（min · 10^6cells） （about 1/30 production of HepG2 cell）. Trypan blue assay showed that the cell viability was greater than 95%, no cytotoxicity by using the present instrument. Conclusion： The modified plate lid was feasible for detection of hydrogen sulfide release in living cells. suggesting that there was instrument in cell culture

关 键 词：硫化氢 细胞 亚甲基蓝 胱硫醚Β合酶 胱硫醚γ裂解酶 

分 类 号：R135.14[医药卫生—劳动卫生]