荧光探针法检测脏器硫化氢方法的建立及验证  

Establishment and verification of method for detection of hydrogen sulfide in organs by fluorescent probe method

作　　者：李娜[1,2] 樊修和 王征[1] 胡伟 杨浩东 熊洋洋 于金高 LI Na;FAN Xiu-he;WANG Zheng;HU Wei;YANG Hao-dong;XIONG Yang-yang;YU Jin-gao(Co-construction Collaborative Innovation Center for Chinese Medicine Resources Industrialization by Shaanxi&Education Ministry/State Key Laboratory of Research&Development of Characteristic Qin Medicine Resources(Cultivation)/Shaanxi Innovative Drug Research Center,Shaanxi University of Chinese Medicine,Shaanxi University of Chinese Medicine,Xianyang,Shaanxi 712046,China;Key Laboratory of Pharmacodynamic Mechanism and Material Basis of Traditional Chinese Medicine,Shaanxi Provincial Administration of Traditional Chinese Medicine,Shaanxi University of Chinese Medicine,Xianyang,Shaanxi 712046,China)

机构地区：[1]陕西中医药大学陕西中药资源产业化省部共建协同创新中心,秦药特色资源研究开发国家重点实验室(培育),陕西省创新药物研究中心,陕西咸阳712046 [2]陕西中医药大学陕西省中医药管理局中药药效机制与物质基础重点研究室,陕西咸阳712046

出　　处：《中国药理学通报》2023年第8期1592-1598,共7页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 82003964);陕西中医药大学校级科研课题(No CXSJ202334)。

摘　　要：目的快速制备并分离纯化硫化氢特异性荧光探针(Washington State Probe-5),建立动物组织中硫化氢的荧光探针测定法,并在癌性胸水模型中进行方法适用性验证。方法优化条件分离纯化WSP-5;对荧光探针反应液配制溶剂、DMSO加入体积、pH、反应液溶剂和反应液体积、样品前处理温度、研磨次数、研磨后静置时间等条件进行考察;以S-180腹水瘤细胞建立癌性胸水小鼠模型,测定小鼠各脏器中的硫化氢含量。结果以硅胶和葡聚糖凝胶为固定相,二氯甲烷-甲醇-甲酸(60∶1∶0.1,V/V/V)和二氯甲烷-甲醇(1∶1,V/V)为洗脱剂制备WSP-5纯品;动物组织样本和硫氢化钠标准溶液加入优化后的5倍量冰冷反应液,低温研磨、高速离心,上清液避光孵育12 h,测定荧光强度并计算硫化氢浓度;该方法检测限约0.6μmol·L^(-1),定量限1μmol·L^(-1),在硫氢化钠一定浓度范围内,浓度与荧光强度线性系数均大于0.99;在癌性胸水小鼠模型中,各脏器硫化氢含量随着注射癌细胞数量的增多均有不同程度的增加趋势。结论该方法操作简单、成本低、灵敏度高、特异性强,适合各类组织样本的高通量检测,可为硫化氢相关药效机制的深入研究和相关药物开发提供方法学参考。Aim To rapidly prepare and purify hydrogen sulfide specific fluorescent probe(WSP-5),establish and optimize the fluorescent probe method for the determination of hydrogen sulfide in animal tissues,and verify the applicability of the method in the model of malignant pleural effusion.Methods The preparation solvent of fluorescent probe reaction solution,DMSO addition volume,pH,reaction solution solvent and reaction solution volume,sample pretreatment temperature,grinding times,and standing time after grinding were investigated.The mouses model of malignant pleural effusion was established with S-180 ascites tumor cells,and the concentration of hydrogen sulfide in various organs and tissues of the model animal was measured.Results After optimization,silica gel and dextran gel were used as stationary phases,dichloromethane methanol formic acid(60∶1∶0.1,V/V/V)and dichloromethane methanol(1∶1,V/V)were used as eluents for separation and purification,and the first eluting component was taken to prepare WSP-5 with a purity of more than 700 mg.Animal tissue samples and sodium hydrosulfide standard solution were added with 5 times of cold reaction solution,after low temperature vibration grinding,high-speed centrifugation,the supernatant was incubated in dark for 12 hours,the fluorescence intensity was measured by fluorescent microplate reader.Hydrogen sulfide concentration was calculated according to the standard curve.The LOD of this method was about 0.6μmol·L^(-1),LOQ 1μmol·L^(-1).Within a certain concentration range of sodium hydrosulfide,the linear coefficients of concentration and fluorescence intensity were greater than 0.99.In the mouses model of malignant pleural effusion caused by S-180 cells,the content of hydrogen sulfide in each organ increased with the increase of the number of cancer cells injected.Conclusions The method established in this study is simple to operate with low cost,high sensitivity and specificity,high-throughput,and it can detect a variety of tissue samples.It can also provide a met

关 键 词：硫化氢 检测方法 荧光探针 WSP-5 方法优化 癌性胸水 

分 类 号：R-332[医药卫生] R194.3R394R73R981.9