人精子间期核的双色荧光原位杂交

Dual-color fluorescence in situ hybridization of human sperm interphase nucleus

目的 建立人精子间期核双色荧光原位杂交 (dual colorfluorescenceinsituhybridization ,D FISH)的实验方法。方法 采用双色荧光原位杂交技术对 12例正常精子标本进行间期核的原位杂交 ,并统计精子间期核X、Y染色体的杂交信号颗粒数量。结果 在显微镜下可见精子头部有以Biotin标记的pBamX7探针显示 1个绿色杂交信号为X染色体精子 (X精子 ) ,以Digoxigenin标记的 pY3.4探针显示 1个红色杂交信号为Y染色体精子 (Y精子 ) ;精子头部有 2个荧光杂交信号的精子为染色体数目异常精子 (如XX、XY、YY精子 ) ;间期核背景经DAPI复染显示蓝色 ;统计 12例正常精子标本总共 4 80 0个精子间期核 ,X精子杂交信号阳性率为 4 8.5 6 % ,Y精子杂交信号阳性率为 4 9.73% ,总共统计 2 4 0 0 0个精子 ,3种双体总杂交率为 0 .197%。结论 本法具有荧光杂交信号直观易分辨、短时间内能大量分析精子数量和实验操作相对简便。

Objective To establish an experimental approach to dual color fluorescence in situ hybridization of human sperm interphase nucleus.Methods The technique of dual color fluorscence in situ hybridization was used in 12 cases of normal sperm specimens,and the number of hybridization signals for X?Y chromosome in the sperm interphase nucleus were counted.Results It was seen under the microscope that the Biotin labeled pBamX7 probe showed 1 green hybridization signal was X chromosomal sperm(X sperm),that the Digoxigenin labeled pY3.4 probe showed 1 red hybridization signal was Ychromosomal sperm(Y sperm),and that 2 fluorescence hybridization signals on the sperm head were abnormal counts for chromosomal sperm(XX?XY?Yysperm).Interphase nucleus counter stained with DAPI showed blue.From 12 cases of normal sperm specimens with the total of 4 800 sperm interphase nuclei,the positive rate for X sperm hybridization signals was 48.56% and the positive rate for Y sperm hybridization signals was 49.73%.Among the altogether 24 000 experimented sperm interphase nuclei,the average hybridization rate of the 3 types being 0.197%.Conclusions The merits of this method are the hybridization signals visible and distinguishable,analyzing the quantity of sperm on a large scale,simple performance of experiments and accurate and reliable results.