O'Sullivan Jacintha N, Finley Jennifer C, Risques Rosa-ana, Shen Wen-Tang, Gollahon Katherine A, Moskovitz Alexander H, Gryaznov Sergei, Harley Calvin B, Rabinovitch Peter S
Department of Pathology, University of Washington, Seattle, Washington 98195-7705, USA.
Cytometry A. 2004 Apr;58(2):120-31. doi: 10.1002/cyto.a.20006.
Telomeres are tandem repeated DNA sequences at the ends of every chromosome, which cap, stabilize, and prevent chromosome fusions and instability. Telomere regulation is an important mechanism in cellular proliferation and senescence in normal diploid and neoplastic cells. Quantitative methods to assess telomere lengths are essential to understanding how telomere dynamics play a role in these processes.
Telomere lengths have been conventionally measured using terminal restriction fragment (TRF), quantitative fluorescence in situ hybridization (QFISH), and flow FISH. In this study, we have applied QFISH to measure average telomere lengths in cultured cells and human tissues of the GI tract. Importantly, this method can be used to analyze telomere lengths in sections using confocal microscopy. We describe and compare three image analysis algorithms: a simple pixel histogram calculation of background corrected fluorescence, a telomere spot-finding method, and a background curve subtraction algorithm.
Using normal human diploid fibroblasts (NHDF) either dropped on slides or sectioned after agar embedding, similar telomere length shortening is evident with increasing population doubling levels (PDLs), using peptide nucleic acid (PNA) and an N3'-P5'-phosphoamidate (PA) oligonucleotide probe for all three methods. Validation of these in situ telomere quantification methods showed excellent agreement with the commonly used telomere repeat fragment-Southern blot method. Telomere length reductions can also be demonstrated in tissue sections from histologically normal mucosa from patients with chronic ulcerative colitis (with dysplasia or cancer elsewhere in the colon), in colon adenomas, and in mucosal biopsies from patients with Barrett's esophagus. Both on slides and in tissue sections, the telomere spot-finding method has the greatest variability, while intra- and inter-biopsy variability in telomere length assessment using the other methods is relatively low.
Accurate and reproducible telomere length measurements can be made in tissue sections using QFISH and confocal microscopy. The simplest methods proved the most reliable and make these methods readily accessible to many laboratories. The use of these methods will enhance the ability to measure telomere lengths in tissue samples and aid in the understanding of the role of telomere length in aging and disease.
端粒是位于每条染色体末端的串联重复DNA序列，其起到封端、稳定染色体以及防止染色体融合和不稳定的作用。端粒调控是正常二倍体细胞和肿瘤细胞中细胞增殖与衰老的重要机制。评估端粒长度的定量方法对于理解端粒动态变化在这些过程中如何发挥作用至关重要。
传统上使用末端限制片段（TRF）、定量荧光原位杂交（QFISH）和流式荧光原位杂交来测量端粒长度。在本研究中，我们应用QFISH来测量培养细胞和胃肠道人体组织中的平均端粒长度。重要的是，该方法可用于通过共聚焦显微镜分析切片中的端粒长度。我们描述并比较了三种图像分析算法：背景校正荧光的简单像素直方图计算、端粒斑点查找方法和背景曲线减法算法。
使用滴加在载玻片上或琼脂包埋后切片的正常人二倍体成纤维细胞（NHDF），对于所有三种方法，随着群体倍增水平（PDL）的增加，端粒长度缩短现象明显，使用肽核酸（PNA）和N3'-P5'-磷酰胺寡核苷酸探针。这些原位端粒定量方法的验证显示与常用的端粒重复片段 - Southern印迹法高度一致。在患有慢性溃疡性结肠炎（结肠其他部位有发育异常或癌症）的患者的组织学正常黏膜的组织切片、结肠腺瘤以及巴雷特食管患者的黏膜活检中也可证明端粒长度减少。无论是在载玻片上还是在组织切片中，端粒斑点查找方法的变异性最大，而使用其他方法进行端粒长度评估时，活检内和活检间的变异性相对较低。
使用QFISH和共聚焦显微镜可以在组织切片中进行准确且可重复的端粒长度测量。最简单的方法被证明最可靠，并且使许多实验室都能够轻松使用这些方法。这些方法的使用将增强测量组织样本中端粒长度的能力，并有助于理解端粒长度在衰老和疾病中的作用。
