成像流式细胞仪检测细胞自噬水平研究  

Disscution of autophagy detection by imaging flow cytometer

作　　者：时景仁 郭向华[1] 王珊珊[1] 刘凯[1] 乔录新[1] 张玉林[1] 陈德喜[1] 

机构地区：[1]首都医科大学附属北京佑安医院/北京市肝病研究所,北京100069

出　　处：《国际检验医学杂志》2016年第21期2949-2951,2955,共4页International Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目(81361120401;81371399;81272266);首都卫生发展科研专项项目(首发2014-1-1151);肿瘤侵袭和转移机制研究北京市重点实验室开放课题(2015ZLQX05);北京市肝病研究所科研课题(BJIH-01602)

摘　　要：目的采用新型成像流式细胞仪量化检测细胞自噬水平,完善自噬检测手段。方法用绿色荧光蛋白(GFP)-微管相关蛋白轻链3(LC3)表达质粒转染Huh7细胞和H1299细胞,并进行24h饥饿处理,诱导自噬发生,分别用荧光显微镜拍照、蛋白免疫印迹法和成像流式细胞术3种方法检测细胞自噬水平。用IDEAS软件对成像流式细胞术的细胞自噬结果进行量化分析。结果荧光显微镜显示,Huh7细胞比H1299细胞中的自噬斑点少;蛋白免疫印迹法显示,与H1299细胞比较,Huh7细胞LC3-Ⅱ与LC3-Ⅰ比例较低,但2种方法均不能获得通量统计数据。成像流式细胞仪结果显示细胞自噬图像,2种细胞的GFP-LC3质粒转染效率基本一致(25.2%和27.6%);比较两者发生高水平自噬(LC3斑点数目为6个以上)的细胞比例,Huh7细胞仅为9.0%,显著少于H1299细胞(26.2%)。结论采用新型成像流式细胞仪检测细胞自噬,兼具量化分析和成像的优点,能弥补荧光显微镜和蛋白免疫印迹法的不足,是一种更优秀的自噬检测方法。Objective To detect autophagy levels in the cells by a novel imaging flow cytometer,and to improve the detection methods of autophagy.Methods GFP-LC3 plasmid was transfected into Huh7 or H1299 cells,respectively,followed by starvation for 24 hours in order to induce autophagy.Autophagy levels in the cells were detected by three ways：fluorescence microscopy,western blotting and imaging flow cytometry.Quantified analysis of cellular autophagy data from the imaging flow cytometer was performed by IDEAS software.Results Fluorescence microscopy showed that the numbers of autophagy spot in Huh7 cells were less than that in H1299 cells.The results of western blotting also indicated that compared with H1299 cells,the ratio of LC3-Ⅱ and LC3-Ⅰin Huh7 cells were lower distinctly.Whereas both of two methods couldn′t obtain quantitative data.The imaging flow cytometer not only displayed the autophagic cellular images,but also demonstrated that only 9.0% of Huh7 cells exhibited over 6LC3spots（high autophagic levels）,markedly less than the same population of H1299cells（26.2%）.While GFP-LC3 plasmid transfection rates of the two kind of cells were similar（25.2% and 27.6%）.Conclusion The novel imaging flow cytometer has the advantages of combination of quantitative data analysis and fluorescent imaging as well as could compensate the shortcomings of fluorescence microscopy and western blotting.It is more excellent to detect autophagy by imaging flow cytometer.

关 键 词：自噬 成像流式细胞仪 微管相关蛋白轻链3 

分 类 号：R446.8[医药卫生—诊断学]