应用流式细胞术检测人牙髓细胞内活性氧的方法探讨  

Detection of reactive oxygen species of human dental pulp cells by flow cytometry

作　　者：伍甜甜[1] 杜嵘[1] 李莉芬[1] 朱亚琴[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院·口腔医学院口腔综合科.上海市口腔医学重点实验室,上海200011

出　　处：《上海口腔医学》2012年第6期617-621,共5页Shanghai Journal of Stomatology

基　　金：国家自然科学基金(81271134);上海科学技术委员会资助项目(08DZ2271100,08JC1414500)~~

摘　　要：目的:建立用流式细胞术检测人牙髓细胞内活性氧变化的方法。方法:采用改良组织块法培养人牙髓细胞,牙髓细胞经不同浓度H2O2处理30 min后,以终浓度分别为10、20μmol/L的2’,7’-二氯荧光素二乙酸酯(2’,7’-dichlorofluoresin diacetate,DCFH-DA)作为荧光探针,与牙髓细胞共同避光孵育20 min,使用流式细胞仪检测细胞内氧化性二氯荧光素(Dichlorofluorescein,DCF)的荧光强度。采用SPSS13.0软件包对数据进行统计学分析。结果:不同浓度探针装载率不同;不同浓度H2O2作用于细胞一定时间后,使用流式细胞术可检测到细胞内活性氧水平产生相应变化,且多次重复结果稳定。结论:选用终浓度为20μmol/L的DCFH-DA作为探针装载率较高,利用流式细胞仪检测人牙髓细胞内活性氧的产生是一种可靠、稳定、简便的方法。PURPOSE： To establish a method for detection of reactive oxygen species （ROS） of human dental pulp cells （HDPCs） by flow cytometry. METHODS： HDPCs were obtained using tissue explant technique in vitro. The subcultured cells were exposed to peroxide oxygen （H202） of different concentrations from 50 μmol/L to 400 μmol/L for 30 minutes, then incubated with two different concentrations of 2＇,7＇-dichlorofluorescein diacetate （DCFH-DA）, which were 10 μmol/ L and 20 μmol/L for 20 minutes at 37C in dark. The fluorescence intensities of intracellular dichlorofluoreseein（DCF） were detected by flow cytometry. Statistical analysis was carried out by SPSS 13.0 software software. RESULTS： The positive rate varied with different concentrations of detectors. The fluorescence intensities remained insignificant difference among samples incubated with the same concentration of detector and H202,and increased by rising of the incubating concentration of H20〉 CONCLUSIONS： The detector with concentration of 20 μmo/L shows higher detector loading rate （positive rate）. The intracellular ROS level changes as the H202 treatment concentration rising from 50 to 400 μmol/L. The application of flow cytometry to measure the ROS in HDPCs is simple, reliable and stable. Supported by National Natural Science Foundation of China （81271134） and Research Fund of Science and Technology Commission of Shanghai Municipality （08DZ2271100,08JC1414500）.

关 键 词：流式细胞术 人牙髓细胞 活性氧 

分 类 号：R780.2[医药卫生—口腔医学]