多色荧光原位杂交方法同时检测人精子染色体数目畸变和结构畸变

Simultaneous Detection of the Numerical and Structural Chromosome Aberrations of Sperm in Normal Men by Multi-color Fluorescence in situ Hybridization (FISH)

背景与目的:建立可以同时检测人精子染色体数目畸变和结构畸变的多色荧光原位杂交技术。材料与方法:使用2条1号染色体探针(着丝粒和末端探针),2条18号染色体着丝粒探针,分别用地高辛或生物素标记,与人精子核DNA进行荧光原位杂交,用CY3-链亲和素检测生物素探针杂交信号;用与FITC结合的抗地高辛抗体检测地高辛信号,结果:在Nikon荧光显微镜下,可以清楚看到精子头部呈现兰色的荧光信号背景下,有红色荧光点信号(1号染色体着丝粒),绿色荧光信号(1号染色体末端),和黄色荧光信号(18号染色体着丝粒部位红、绿两种荧光信号的混合色)。本方法能够同时检测到精子1号,18号染色体非整倍体率(双体率,缺体率),1号染色体短臂和着丝粒结构畸变(重复率,缺失率),以及二倍体精子率3种染色体异常。用建立的方法检测14名正常人135 937条精子,测得1号染色体双体率、缺体率分别为O.045％和0.048％;18号染色双体率、缺体率分别为0.053％和O.045％:二倍体精子率为0.061％;1号染色体末端重复率、缺失率分别为0.082％、0.069％,1号着丝粒重复率、缺失率分别为O.075％,O.060％;均在文献报道范围内。结论:本研究建立的多色FISH可用于测定正常人和环境化学物接触人群精子染色体数目畸变和结构畸变。

BACKGROUND & AIM: The purpose of this paper was to develop a method of multi - color FISH which could be used to detect the frequencies of numerical and structural chromosomal aberrations in sperm simultaneously. MATERIAL AND METHODS:Two probes for chromosome 1(one for centromere labeled with biotin,the other for terminal of short arm labeled with digoxingenin) and two probes for centromere chromosome 18 labeled with biotin or digoxingenin. The hybridization signals were detected with CY3- strepavidin for botin - labeled probes, and anti -digoxingenin antibodies conjugated with FITC for digoxingenin probes. RESULTS: Under the blue fluorescenses background , three hybridized signals red , green, and yellow , were clearly visible in the sperm nucleus . The red signals represented the target of chromosome 1 ; green signals represented the target of chromosome lp terminal, and yellow signals (which was the mixed color of red and green) represented the centromere of chromosome 18. Three kinds of chromosomal abnormalities were simultaneously detected by the multi - color FISH: the frequencies of disomic and nullisomic sperm for chromosome 1 and 18; the frequencies of duplication and deletion of cetromere , telemere for chromosome 1; and the frequency of diploidy sperms. A total of 135 937 sperm nucleus of 14 normal health men were counted. The frequencies of numerical and structural aberrations were detected. The frequencies of disomic and nullisomic were 0.045 % and 0.048 % for chromosome 1, 0.053 % and 0.045 % for chromosome 18, respectively; the frequencies of duplication and deletion for 1p terminal were 0.082 % and 0.069 % respectively; the frequencies of duplication and deletions for chromosome 1 centromere were 0.075 % and 0.06 0 % respectively; and the frequencies of diploidy sperm was 0.061 %. All the values detected were in the range of the data published in literature . CONCLUSION: Multi - color FISH could be used to detect the numerical and structural chromosome aberrations of sperm in normal men and exposed to environmental chemicals.