Ferlicot Sophie, Youssef Nelly, Feneux Danielle, Delhommeau François, Paradis Valérie, Bedossa Pierre
Services d'Anatomie Pathologique, Hôpital de Bicêtre, 78 Rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France.
J Pathol. 2003 Aug;200(5):661-6. doi: 10.1002/path.1392.
Loss of telomere repeat sequences occurs after each cell division and telomere shortening has been implicated in cellular senescence. The measurement of telomere length might therefore assess the lifespan of a cell. The aim of this study was to set up and validate a technique enabling the assessment of telomere length on tissue sections. Quantitative fluorescence in situ hybridization (Q-FISH) with telomeric probes was performed on smears and sections from cell preparations or human tissues. The mean fluorescence intensity of telomere spots (FI/spot) was automatically quantified by image analysis. Telomeric restriction fragment (TRF) length was assessed by Southern blotting. There was a positive significant correlation between telomere length, as assessed by Q-FISH, and TRF length determined by Southern blotting in corresponding samples (p < 0.01, r = 0.6 for tissue and p < 0.01, r = 0.79 for cells). FI/spot was higher on smears than on sections, but pairwise comparison showed a significant correlation both for cells and for tissues (r = 0.77, p < 0.001 for cells and p < or = 0.01, r = 0.64 for tissue). Finally, since telomere length is expected to shorten with age, FI/spot was assessed in liver samples according to the age of patients: a negative correlation was demonstrated (r = 0.76, p < 0.01). Inter-assay variation was 7% for Q-FISH performed on tissue sections and 12% on touch preparations. This study shows that Q-FISH can be performed with confidence on fixed frozen tissue sections in order to assess telomere length. It is an easy, accurate, and reproducible in situ method for assessing telomeres in the context of cell type and tissue architecture.
每次细胞分裂后都会出现端粒重复序列的丢失，端粒缩短与细胞衰老有关。因此，端粒长度的测量可能会评估细胞的寿命。本研究的目的是建立并验证一种能够在组织切片上评估端粒长度的技术。使用端粒探针进行定量荧光原位杂交（Q-FISH），对细胞制剂或人体组织的涂片和切片进行检测。通过图像分析自动定量端粒斑点的平均荧光强度（FI/斑点）。通过Southern印迹法评估端粒限制性片段（TRF）的长度。在相应样本中，通过Q-FISH评估的端粒长度与通过Southern印迹法测定的TRF长度之间存在显著正相关（组织样本中p < 0.01，r = 0.6；细胞样本中p < 0.01，r = 0.79）。涂片上的FI/斑点高于切片，但成对比较显示细胞和组织样本中均存在显著相关性（细胞样本中r = 0.77，p < 0.001；组织样本中p ≤ 0.01，r = 0.64）。最后，由于预期端粒长度会随着年龄增长而缩短，因此根据患者年龄对肝脏样本中的FI/斑点进行了评估：结果显示存在负相关（r = 0.76，p < 0.01）。对组织切片进行Q-FISH检测时，批间变异为7%，对触摸涂片进行检测时批间变异为12%。本研究表明，可以在固定的冷冻组织切片上可靠地进行Q-FISH检测，以评估端粒长度。它是一种在细胞类型和组织结构背景下评估端粒的简便、准确且可重复的原位方法。
