Negoescu A, Guillermet C, Lorimier P, Brambilla E, Labat-Moleur F
Laboratoire de Pathologie Cellulaire, CHRU, BP 217X, Grenoble, France.
Biomed Pharmacother. 1998;52(6):252-8. doi: 10.1016/S0753-3322(98)80010-3.
In the absence of a universal specific molecular tracer of apoptosis, structural DNA alterations provide the basis of labeling systems: double-strand fragmentation for TUNEL (terminal transferase-mediated dUTP nick end-labeling), denaturation for poly (A) in situ hybridization, immunogenicity of single strand DNA, all methods which imply limited specificity due to the unavoidable presence of DNA breaks in virtually all cells. Thus, TUNEL application has been restrained to a narrow spectrum of sample conditions which has limited, in particular, retrospective surveys and apoptotic nuclei-protein double labelings. In the apoptotic nucleus two main obstacles intervene between TUNEL reagents and their targets: DNA hypercondensation and proteins around DNA. The former increases in the course of apoptosis and both are worsened by crosslinking and precipitating fixatives. This point out that TUNEL is an ambitious approach whose target, apoptotic DNA breaks, is less accessible than breaks occurring in non-apoptotic less compacted DNA. However, TUNEL has an advantage: the far greater degree of apoptotic DNA fragmentation. How to obtain a frank differential staining between apoptotic and non-apoptotic DNA? It appears that the answer relies on the pretreatment step and not in modifying the TUNEL staining protocol, which is optimal. Adapted pretreatments are able to circumvent accessibility obstacles and to extend TUNEL applicability to the most demanding conditions, those of archived tissue samples and of TUNEL--protein double labelings.
在缺乏凋亡通用特异性分子示踪剂的情况下，结构性DNA改变为标记系统提供了基础：用于TUNEL（末端转移酶介导的dUTP缺口末端标记）的双链断裂、用于多聚（A）原位杂交的变性、单链DNA的免疫原性，所有这些方法由于几乎所有细胞中不可避免地存在DNA断裂而意味着特异性有限。因此，TUNEL的应用局限于狭窄的样本条件范围，这尤其限制了回顾性研究以及凋亡细胞核-蛋白双重标记。在凋亡细胞核中，TUNEL试剂与其靶标之间存在两个主要障碍：DNA高度浓缩和DNA周围的蛋白质。前者在凋亡过程中增加，并且两者都会因交联和沉淀固定剂而恶化。这表明TUNEL是一种具有挑战性的方法，其靶标——凋亡DNA断裂，比非凋亡状态下结构较松散的DNA中出现的断裂更难接近。然而，TUNEL有一个优势：凋亡DNA片段化程度要高得多。如何在凋亡DNA和非凋亡DNA之间实现明显的差异染色？答案似乎在于预处理步骤，而不是修改TUNEL染色方案，因为该方案已经是最优的。合适的预处理能够克服可及性障碍，并将TUNEL的适用性扩展到最苛刻的条件，即存档组织样本和TUNEL-蛋白双重标记的条件。
