活细胞硫化氢检测新方法.pdf

化京大学学报
医学版
JOURNAL OF PEKING UNIVERSITY(HEALTH SCIENCES)Vol45 No.3 Jun 2013
技术方法
活细胞硫化氬檢测新方法
谢静2,曾强,郑扬,廖锋2,徐国恒,唐朝枢2,耿彬2
(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)
[摘要]的:建立测定活细胞释放微量硫化氢( hydrogen sulfide,H12S)的简易方法。方広: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HepC2细胞和人脐静脉内皮细胞硫化氢的释放量。結累:HepC2细胞添加
L-半胱氨酸以及辅酶磷酸吡哆醛后,继续培养12h,H2S释放量为(859.39士19.12)mmol/(min:10°cel);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:10cel);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)mmo/(min:10°cels);
两种酶抑制剂联合使用后H2S释放量显著抑制,为(204.47±97.14)mol/(min:10°cels)。人脐静脉内皮细胞也
可内源性产生H2S, HUVEC细胞H1S的释放量为(26.23±3.24)mol/(min?10°cels),约为HepC2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。結恰: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。
[关键词]硫化氢;细胞;亚甲基蓝;胱硫瞇β合酶;胱硫γ裂解酶
中图分类号]R33-33[文献标志码]A[文章编号]1671-167X(2013)03-0489-04
doi:10.3969/j.is.1671-167X.2013.03.029
A new methods for determining hydrogen sulfide release in cultured cells
XIE Jing", ZENG Qiang, ZHENG Yang, LIAO Feng, XU Guo-heng, TANG Chao-shu, GENG Bin
(1. International Medical Center, PLA General Hospital, Beijing 100853, China; Department of Physiology and Patho-
physiology, Peking University Health Science Center, Beijing 100191, China)
ABSTRACT Objective To establish a simple method of measuring hydrogen sulfide(H 5)in cultured
living cells. Methods Fi
membrane was stuck on the lid of cell culture plate. H,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 H, S production from the living cells was
counted according to the standard curve. This simple method was used to access the H S release in
EPG2 (high expression CBS and CSE )and HUVEC (low expression CSE) cell lines. Results
generation in cultured Hepg2 cells assayed using the present method was(859. 39 *19. 12 )nmol/
(min 10 cells ) PAG(CSE inhibitor), HA(CBS inhibitor) or the two-inhibitor(PAG+HA treat-
ment significantly lowered H,S release, respectively: (341. 34 *105 90) nmol/(min. cells
(375.05±174.50)mmol/(min·10°cels),and(204.47±97.14)mmol/(min:10°cl).TheH
roductor
of HUVEC was(26. 23=#3. 24) nmol/(min. 10cells)(about 1/30 production of Hep
Trypan blue assay showed that the cell viability was greater than 95%0, suggesting that there was
no cytotoxicity by using the
present instrument. Conclusion: The modified instrument in cell culture
plate lid was feasible for detection of hydrogen sulfide release in living cells
KEY WORDS Hydrogen sulfide; Cells; Methylene blue; Cystathionine gamma-lyase; Cystathionine
beta
内源性硫化氦( hydrogen sulfide,H2S)是继 NO thase,CBS)、胱硫醚γ-裂解酶( cystathionine-y-
和CO之后发现的又一种重要的气体信号分子, lyase,CSE)和巯基丙酮酸转硫酶(3- mercapto
具有多种生物学效应,参与多系统、多种疾病的病
vate transsulphurase,MPST)。应用离体培养细胞
理功能调节2。内源性H2S在体内主要由3种酶来进行观察是医学研究重要的手段,灵敏、准确地
催化产生:胱硫醚-β-合酶( cystathionine-β-syn-测量在培养细胞H12S的释放量时十分关键,传统
基金项目:国家自然科学基金(81170235)和“十二五”国家科技支撑计划(2012BAIB7BO4)项目资助 Supported by the National Natural Sci-
ence Foundation of China(81170235) and the National Scienee and Technology Pillar Program during the 12th Five-year Plan(2012BA137B04)
ACorrespondingauthor"se-mail,bingen@hse.pku.edu.cn
网络出版时间:2013-4-258: