地高辛标记法检测人源细胞系端粒长度

A Digoxin-Labelled Method to Measure the Length of Telomere Restriction fragment of Human Chromosome

目的:建立一种灵敏的、非同位素标记的端粒长度检测方法,并用于人源细胞系的肿瘤研究。方法:以地高辛标记寡核苷酸(TTAGGG)3为探针,对基因组DNA进行Southern印迹检测,经CDP-Star显色后与DNA标准分子量比较,利用图像分析系统计算端粒长度。结果:通过严格控制探针的工作浓度(1∶3000稀释)、DNA上样量(1.5～2.5μg)、杂交温度(42±1℃)等主要影响因素,获得了比较好的杂交条带。结论:建立了一种稳定、可靠、低背景的地高辛标记检测端粒长度的方法;对正常细胞、永生化细胞和肿瘤细胞进行了端粒长度的检测和对比分析。

Objective： To measure telomere length （length of telomere restriction fragment, TRF） by hybridization with non-radio-labeled probes and study its applicaion in cancer research of human. Methods： Telomere length was examined by Southern blotting by using labelled oligonucleotide （TTAGGG）3 probe with digoxin. After Southern hybridization, the blot was stained with CDP-Star, analysed by using a densitometric imaging system and mean length of TRF calculated, comparing with standard kilobase sized marker. Results： The hybridization bands were showed under stringently controlling the probe working concent ration （1：3 000 dilution）, loaded DNA sample （1.5-2.5μg） and hybridization temperature （42±1℃）. Conclusion： A digoxin-labelled, stable, reliable and low background method for determining the telomere length of human chromosome is established. Meanwhile, the telomere length of normal cells, immortalized cells and tumour cells have been analysed with this method.