Quantification of dystrophin immunofluorescence in dystrophinopathy muscle specimens
Quantification of dystrophin immunofluorescence in dystrophinopathy muscle specimens
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DOI：
10.1111/j.1365-2990.2012.01250.x
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发表时间：
2012-10-01
影响因子：
5
通讯作者：
Flanigan, K. M.
中科院分区：
文献类型：
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作者：
Taylor, L. E.;Kaminoh, Y. J.;Flanigan, K. M.
L.E.Taylor，Y.J.Kaminoh，C.K.Rodesch和K.M.Flanigan(2012年)神经病理学和应用神经生物学38,591601肌营养不良症肌肉标本中dystrophin免疫荧光的定量研究目的：Duchenne肌营养不良症通常与肌膜肌营养不良蛋白表达缺失或几乎缺失有关。在肌肉活组织切片的免疫荧光研究中，对极低水平的肌营养不良蛋白信号进行量化是一项技术挑战。这在原则验证药物试验的环境中尤其如此，在这种试验中，即使营养不良蛋白的绝对水平仍然很低，检测和量化可能显著的表达水平变化也是重要的。方法：我们开发了一种图像分析方法，可以可靠和半自动地定量检测抗肌营养不良蛋白在共染色切片中的低水平表达。使用自定义变形脚本来创建连续区域的血影蛋白掩膜，我们仅在血影蛋白掩膜内可能代表肌膜的像素处量化抗肌营养不良蛋白的信号强度。利用这种方法，我们分析了一系列DMD、Becker肌营养不良症、中间型肌营养不良症患者的肌肉活检组织和正常对照组织。结果：对多天连续切片的分析证实了其重复性，归一化的dystrophin/Spectrin强度比(表示为正常对照组织的百分比)与Western印迹分析确定的dystrophin表达水平有很好的相关性。结论：该方法为DMD治疗试验提供了一种可靠可靠的生物标志物检测方法。
L. E. Taylor, Y. J. Kaminoh, C. K. Rodesch and K. M. Flanigan (2012) Neuropathology and Applied Neurobiology38, 591601 Quantification of dystrophin immunofluorescence in dystrophinopathy muscle specimens Aims: Duchenne muscular dystrophy (DMD) is usually associated with absent or nearly absent dystrophin expression at the sarcolemmal membrane. Quantification of very low levels of dystrophin signal in immunofluorescent studies of muscle biopsy sections presents a technical challenge. This is particularly true in the setting of proof-of-principle drug trials, in which the detection and quantification of what may be significant changes in levels of expression is important, even if absolute dystrophin levels remain low. Methods: We have developed a method of image analysis that allows reliable and semi-automated immunofluorescent quantification of low-level dystrophin expression in sections co-stained for spectrin. Using a custom Metamorph script to create a contiguous region spectrin mask, we quantify dystrophin signal intensity only at pixels within the spectrin mask that presumably represent the sarcolemmal membrane. Using this method, we analysed muscle biopsy tissue from a series of patients with DMD, Becker muscular dystrophy, intermediate muscular dystrophy and normal control tissue. Results: Analysis of serial sections on multiple days confirms reproducibility, and normalized dystrophin : spectrin intensity ratios (expressed as a percentage of normal control tissue) correlate well with the dystrophin expression levels as determined by Western blot analysis. Conclusion: This method offers a robust and reliable method of biomarker detection for trials of DMD therapies.