基于5-乙炔基-2'-脱氧尿苷渗入法的不同细胞增殖检测方法比较

Comparison of different methods for cell proliferation measurement based on 5-ethynyl-2'-deoxyuridine incorporation

【目的】基于5-乙炔基-2'-脱氧尿苷(5-ethynyl-2'-deoxyuridine,Ed U)掺入法,比较利用荧光成像和流式细胞术检测细胞增殖的两种方法。【方法】细胞株采用小鼠巨噬细胞系RAW264.7,在六孔细胞培养板中用盖玻片爬片,Ed U标记后,细胞爬片用荧光成像法,其余细胞用流式细胞术分别检测细胞增殖率。应用Bland-Altman法检测两种方法测量细胞增殖率的一致性,组内相关系数(interclass correlation coefficient,ICC)检测两种方法的重复性。【结果】荧光显微镜下观察呈现明亮绿色荧光的细胞为Ed U标记的处于增殖状态的RAW264.7细胞,其余无绿色荧光的为非增殖期细胞。荧光成像半定量分析的结果显示,约(46.75±16.87)%的细胞处于增殖期;流式细胞术定量结果显示(49.50±3.74)%的细胞处于增殖期,与荧光成像半定量结果比较,未见统计学显著性(P=0.4120)。Bland-Altman一致性检验表明,96.30%的点落在95%一致性区间内,偏倚为-2.750;重复性检测表明荧光成像法所得细胞增殖率的组内相关系数为0.562,小于0.75;流式细胞术所得细胞增殖率的组内相关系数为0.869,大于0.75。【结论】荧光成像法和流式细胞术分析均可用于Ed U标记法检测细胞增殖,与荧光成像相比,流式细胞术分析法操作简便、重复性好。

【Objective】To compare the methods for detection of cell proliferation using 5-ethynyl-2′-deoxyuridine（Ed U） incorporation by immunofluorescence and flow cytometry.【Methods】RAW264.7 cells were seeded on glasses and labeled with Ed U. Then the efficacy for the detection of Ed U-positive cells were determined simultaneously by immunofluorescent microscopy and flow cytmetry, respectively. The agreement between these two methods was analyzed by Bland-Altman plot, and the repeatability of measured data was analyzed by intraclass correlation coefficient.【Results】 Ed U positive RAW264.7 cells presented green fluorescence, and about（46.75±16.87） % cells were in proliferating phase according to the microscopy-based semi-quantitative analysis. Flow cytometry analysis of RAW264.7 cells showed that（49.50±3.74）% cells were proliferating cells, with no statistical significance compared with those measured by microscopy by paired t test（P=0.4120）. Bland-Altman diagram shown 96.3% of the data were within the 95% limits of agreement with the bias of-2.750. Intraclass correlation coefficient analysis showed that the microscopy-based method yielded a coefficient of 0.562, whereas flow cytometry-based method could yield a coefficient as high as 0.869.【Conclusion】For the detection of cell proliferative activity using Ed U incorporation, flow cytometry-based method yields higher reproducibility compared with microscopybased semi-quantitative method.