李鹏昊，曲　婷，黄继华，韩婷婷，温子娜，崔淑艳，钟　影

一种稳定的人卵母细胞染色体荧光原位杂交技术

李鹏昊，曲婷，黄继华，韩婷婷，温子娜，崔淑艳，钟影*

（成都市锦欣生殖医学与遗传学研究所，成都市锦江区妇幼保健院，四川成都610066）

目的： 建立一种稳定、简便的人卵母细胞染色体荧光原位杂交技术，为揭示母方减数分裂错误及其发生原因、探讨非整倍性发生机制、评价外环境因素对人卵母细胞的遗传学效应提供有力工具。方法：经签订知情同意书后，取体外受精术后废弃的人卵母细胞，经低渗、渐进固定处理、制备人卵母细胞染色体核型。应用人16号染色体着丝粒荧光探针，经变性处理后与人卵母细胞核型进行荧光原位杂交，再经洗脱和染色等步骤，在荧光显微镜下分析。结果：经过上百批次重复实验，人卵母细胞染色体形态与分散良好，荧光信号明亮清晰、背景干净。结论：成功建立人卵母细胞染色体荧光原位杂交技术。

人卵母细胞；染色体；荧光原位杂交；减数分裂错误；非整倍性

【ABSTRACT】OBJECTIVE: In humans，aneuploidies occur in at least 5% of all clinically recognized pregnancies，among which about 90% can be caused by maternal meiotic error. In this study，a stable and simple method for fluorescence in situ hybridization (FISH) with human oocyte chromosomes was developed to explore causes of maternal meiotic error，mechanisms of aneuploidy and genetic effects of environmental factors on human oocytes. METHODS：The abandoned human oocytes were collected from IVF patients who signed consent forms. The oocytes were used to prepare chromosomal karyotype through hypotonic and gradual fixative treatments. Human oocyte metaphase FISH was performed using the CEP 16 Probe. RESULTS：From our observations，the morphology and dispersal of human oocyte chromosomes were excellent，and the fluorescence signals were bright and clear with clean background. CONCLUSION：We established successfully the method for FISH with human oocyte chromosomes.

【KEY WORDS】human oocyte；chromosome；fluorescence in situ hybridization；meiosis error；aneuploidy

人类精、卵的细胞遗传学研究具有重大意义，因为精、卵的任何染色体异常都会对下一代造成严重后果。自1978年Rudak建立人精子染色体制备技术以来[1]，人精细胞遗传学研究发展很快，而人卵细胞遗传学研究由于实验材料获得困难，进展非常缓慢。荧光原位杂交(fluorescence in situ hybridization，FISH)技术是利用荧光标记探针与染色体上的靶DNA序列杂交，通过检测荧光信号，从而对目的片段进行定性、定量或相对定位的分子细胞遗传学技术，具有特异性强、敏感率高、检测时间短、直观性好等优点，将其与人卵母细胞染色体制备技术相结合，检测人卵母细胞染色体异常，具有广阔的应用前景[2]。……