使用Image-J软件对暴露于（111）铟-二乙三胺五乙酸-人表皮生长因子（In-DTPA-hEGF）或γ射线的乳腺癌细胞中γ-H2AX焦点的数量、面积和密度进行计算分析。

Computational analysis of the number, area and density of gamma-H2AX foci in breast cancer cells exposed to (111)In-DTPA-hEGF or gamma-rays using Image-J software.

作者信息

Cai Zhongli, Vallis Katherine A, Reilly Raymond M

机构信息

Department of Pharmaceutical Sciences, University of Toronto, Ontorio, Canada.

出版信息

Int J Radiat Biol. 2009 Mar;85(3):262-71. doi: 10.1080/09553000902748757.

DOI:10.1080/09553000902748757

PMID:19296344

Abstract

PURPOSE

To develop a simple method for the quantification of gamma-H2AX focus number, density and size.

METHODS

MDA-MB-468 human breast cancer cells were treated overnight with (111)In-diethylenetriaminepentaacetic acid human epidermal growth factor ((111)In-DTPA-hEGF, 0-142 kBq/pmol) or exposed to gamma-radiation to induce DNA double strand breaks (DSB). DNA DSB formation was evaluated by detection of phosphorylated histone H2AX on serine 139 (gamma-H2AX) using immunofluorescence. Confocal microscopy was used to capture images of gamma-H2AX foci and cell nuclei. Image-J software with customized macros was used to quantify gamma-H2AX foci.

RESULTS

The number of gamma-H2AX foci per nucleus scored using Image-J correlated strongly with the number scored using direct visual confirmation (coefficient of determination, R(2) = 0.950; 60 nuclei scored). The mean density (grayscale values per pixel), area and integrated density (IntDen) of individual foci increased linearly as the specific radioactivity (SR) increased up to 67 kBq/pmol (R(2) values of 0.826, 0.964, 0.978, respectively). The mean number of foci per nucleus, the combined area of gamma-H2AX foci per nucleus and the IntDen per nucleus also increased linearly with SR, giving R(2) values of 0.926, 0.974 and 0.983, respectively. Similar linear relationships were observed with the gamma-ray absorbed dose up to 3.0 Gy.

CONCLUSIONS

The density, area and IntDen of individual foci, as well as the number of gamma-H2AX foci, total focus area and IntDen per nucleus were successfully quantified using Image-J with customized macros.

摘要

目的

开发一种简单的方法来定量γ-H2AX焦点的数量、密度和大小。

方法

用（111）铟-二乙三胺五乙酸人表皮生长因子（（111）In-DTPA-hEGF，0-142 kBq/pmol）处理MDA-MB-468人乳腺癌细胞过夜，或使其暴露于γ射线以诱导DNA双链断裂（DSB）。通过免疫荧光检测丝氨酸139位点磷酸化的组蛋白H2AX（γ-H2AX）来评估DNA DSB的形成。使用共聚焦显微镜捕获γ-H2AX焦点和细胞核的图像。使用带有定制宏的Image-J软件对γ-H2AX焦点进行定量。

结果

使用Image-J对每个细胞核中γ-H2AX焦点数量的评分与直接目视确认的评分高度相关（决定系数，R² = 0.950；对60个细胞核进行了评分）。随着比活度（SR）增加至67 kBq/pmol，单个焦点的平均密度（每像素灰度值）、面积和积分密度（IntDen）呈线性增加（R²值分别为0.826、0.964、0.978）。每个细胞核的平均焦点数量、每个细胞核中γ-H2AX焦点的总面积和每个细胞核的IntDen也随SR呈线性增加，R²值分别为0.926、0.974和0.983。在高达3.0 Gy的γ射线吸收剂量下也观察到了类似的线性关系。