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一种神经元轴突变性的量化分析方法

ISSN号：1007-7669
期刊名称：《中国新药与临床杂志》
时间：0
分类：Q189[生物学—神经生物学;生物学—普通生物学]
作者机构：[1]遵义医学院基础药理教育部重点实验室及特色民族药国际合作联合实验室,贵州遵义563099, [2]上海交通大学医学院药学系化学教研室,上海200025, [3]奥林巴斯(中国)有限公司,北京100015
相关基金：国家自然科学基金资助项目（NO：81473201）; 贵州省教育厅“125”重大科技专项（NO：2012012）

关键词：神经元, 轴突变性, AΒ25-35, 变性指数, 免疫荧光, 消卷积, neuron, axonal degeneration, Aβ25-35, degeneration index, immunofluorescence, deconvolution

中文摘要：

目的建立一种神经元轴突变性的量化分析方法。方法采用Aβ25-35（10^-7、10^-6、10^-5mol/L）诱导的大鼠原代海马神经元轴突变性模型,经神经元特异性标记蛋白βⅢTubulin免疫荧光染色获得神经元形态学荧光图像。荧光图像经二维消卷积和二元化处理,将荧光标记的神经元区域转变为黑色并测量其面积,即为神经元总面积。应用Image J软件的颗粒分析功能测量轴突变性产生的片段和颗粒面积,并计算其占神经元总面积的百分比,即可获得神经元轴突变性的量化数据,命名为变性指数（degeneration index,DI）。Western blot测定突触后致密蛋白95（postsynaptic density 95,PSD95）和突触素（synaptophysin,SYN）蛋白变化。Pearson法分析DI与PSD95、SYN变化的相关性。结果免疫荧光染色结果显示Aβ25-35可诱导海马神经元轴突变性,DI显著高于对照组并呈剂量依赖性,PSD95和SYN蛋白水平显著降低,与DI变化呈显著正相关。结论本方法可获得准确的神经元轴突变性的量化数据。

英文摘要：

Objective To establish a quantitative analysis method for axonal degeneration of neurons. Methods The axonal degeneration of the primary hippocampus neurons isolated from the neonatal rat was induced by Aβ25-35（ 10^-7,10^-6,10^-5mol/L）. The profile of neurons was visualized by immunofluorescence staining using mouse monoclonal anti-beta III tubulin antibody. The fluorescence image was manipulated by the two dimensional deconvolution and binarization transformation such that pixel intensity of regions corresponding to neurons were converted to black and all the other regions were converted to white. The total number of black pixel was measured as the total neurons area. The particle analyzer module of Image J software was used to count the area of the small fragments or particles. The percentage of area of the small fragments or particles in total neurons area was calculated,which was named degeneration index（ DI）. Western blot was employed to determine the protein levels of postsynaptic density protein 95（ PSD95） and synaptophysin（ SYN）. Correlation analysis of DI with PSD95 and SYN were executed by Pearson. Results The results of immunofluorescence staining showed that Aβ25-35 treatment led to the significant axonal degeneration of the hippocampus neurons,significantly increased DI with a dose-dependence manner and obviously reduced the protein levels of PSD95 and SYN. There was a significant positive correlation between DI and PSD95 and SYN protein levels. Conclusion This method can be used to obtain accurate quantitative data of axonal degeneration.

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