流式细胞术检测荧光素标记结核杆菌的方法  

The method of detecting fluorescein-labeled tuberculosis by flow cytometry

作　　者：姜丽娜 苏振兴 刘宏升 郑如明 李君如 JIANG Li-Na;SU Zhen-Xing;LIU Hong-Sheng(School of Stomatology,Bengbu Medical University,Bengbu 233030,Anhui,China)

机构地区：[1]蚌埠医科大学口腔医学院,安徽蚌埠233030 [2]蚌埠医科大学基础医学院

出　　处：《吉林医学》2024年第8期1789-1793,共5页Jilin Medical Journal

基　　金：安徽省高等学校自然科学研究重点项目[项目编号:2022AH051482、kj2016A867];安徽省重点实验室、工程中心开放课题[项目编号:BYKL1406ZD]。

摘　　要：目的:建立流式细胞术检测荧光素标记结核杆菌(M.tb)的方法。方法:以异硫氰酸荧光素(FITC)、二乙酸荧光素(FDA)、PKH26和碘化丙啶(PI)标记M.tb,应用流式细胞仪检测M.tb标记率。结果:FITC标记M.tb,在PBS缓冲液中,不同浓度FITC(10~40μg/ml)标记率随浓度增加而增高,FITC浓度10μg/ml标记率为85%,FITC浓度增加到40μg/ml标记率可达到93%,差异具有统计学意义(P<0.05);而在CB缓冲液中,不同浓度FITC(10~40μg/ml)标记M.tb标记率均在90%左右,差异无统计学意义(P>0.05)。FDA标记M.tb标记率均达到92%以上。PKH26标记M.tb标记率均达到90%以上。PI标记M.tb,酒精作用1 min标记率在47%左右,酒精作用2 min标记率达到97%左右,且标记率随时间增加而增高(P<0.05)。结论:建立流式细胞术检测荧光素标记M.tb的方法可为结核病基础研究提供实验思路。创新性建立应用荧光素FITC、FDA、PKH26、PI标记M.tb,并利用流式细胞术检测标记率,可为结核病基础研究提供实验思路。Objective To establish a flow cytometry method for detecting fluorescein-labeled mycobacterium tuberculosis(M.tb).Method M.tb was labeled with Fluorescein Isothiocyanate,Fluorescein Diacetate,PKH26 and Propidium Iodide.The labeling rate of M.tb was detected by flow cytometry.Results M.tb was labeled with FITC,and there were significant differences in the labeling yield of different concentrations of FITC in PBS buffer,the labeling rate of FITC at 10μg/ml was 85%,and the labeling rate of FITC at 40μg/ml was 93%,and the labeling rate increased with the increase of concentration(P<0.05);In CB buffer,the labeling rate of FITC-labeled M.tb with different concentrations was about 90%,and there was no significant difference(P>0.05).The labeling rate of FDA labeled M.tb was more than 92%.The labeling rate of M.tb labeled by PKH26 was more than 90%.The labeling rate of M.tb was 47%after 1min of alcohol treatment,and 97%after 2min of alcohol treatment.The labeling rate increased with time(P<0.05).Conclusion The establishment of a flow cytometry method for the detection of fluorescein-labeled M.tb can provide experimental ideas for the basic research of tuberculosis.

关 键 词：流式细胞仪 荧光素 结核杆菌 

分 类 号：R378.911[医药卫生—病原生物学]