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首页> 外文会议>Conference on Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues II; 20040127-20040128; San Jose,CA; US >Assessment of DNA replication in central nervous system by Laser Scanning Cytometry
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Assessment of DNA replication in central nervous system by Laser Scanning Cytometry

机译：激光扫描细胞仪评估中枢神经系统中DNA复制

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In neurons of patients with Alzheimer's disease (AD) signs of cell cycle re-entry as well as polyploidy have been reported, indicating that the entire or a part of the genome of the neurons is duplicated before its death but mitosis is not initiated so that the cellular DNA content remains tetraploid. It was concluded, that this imbalance is the direct cause of the neuronal loss in AD. Manual counting of polyploidal cells is possible but time consuming and possibly statistically insufficient. The aim of this study was to develop an automated method that detects the neuronal DNA content abnormalities with Laser Scanning Cytometry (LSC).Frozen sections of formalin-fixed brain tissue of AD patients and control subjects were labelled with anti-cyclin B and anti-NeuN antibodies. Immunolabelling was performed using Cy5- and Cy2-conjugated secondary antibodies and biotin streptavidin or tyramid signal amplification. In the end sections of 20 μm thickness were incubated with propidium iodide (PI) (50μg/ml) and covered on slides. For analysis by the LSC PI was used as trigger. Cells identified as neurons by NeuN expression were analyzed for cyclin B expression. Per specimen data of at least 10,000 neurons were acquired. In the frozen brain sections an automated quantification of the amount of nuclear DNA is possible with LSC. The DNA ploidy as well as the cell cycle distribution can be analyzed. A high number of neurons can be scanned and the duration of measuring is shorter than a manual examination. The amount of DNA is sufficiently represented by the PI fluorescence to be able to distinguish between eu- and polyploid neurons.
机译：在患有阿尔茨海默氏病（AD）的患者的神经元中，已有细胞周期重入和多倍体迹象的报道，这表明神经元的全部或部分基因组在死亡之前就已复制，但没有开始有丝分裂，因此细胞DNA含量仍为四倍体。结论是，这种失衡是AD神经元丢失的直接原因。人工计数多倍体细胞是可能的，但是很费时间，而且统计上可能不够。这项研究的目的是开发一种自动方法，通过激光扫描细胞仪（LSC）检测神经元DNA含量异常.AD患者和对照组受试者福尔马林固定的脑组织的冷冻切片用抗细胞周期蛋白B和抗NeuN抗体。使用Cy5和Cy2偶联的二抗和生物素链霉亲和素或金字塔型信号扩增进行免疫振铃。在厚度为20μm的末端切片中，将碘化丙啶（PI）（50μg/ ml）孵育，并盖在玻片上。为了由LSC分析，将PI用作触发器。分析通过NeuN表达鉴定为神经元的细胞的细胞周期蛋白B表达。每个标本至少采集了10,000个神经元的数据。在冷冻的大脑切片中，使用LSC可以自动定量核DNA的数量。可以分析DNA倍性以及细胞周期分布。可以扫描大量神经元，并且测量时间比手动检查要短。 PI荧光足以代表DNA的量，从而能够区分正常和多倍体神经元。

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