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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、结果准确可信等优点
Objective To establish an experimental method for dual color fluorescence in situ hybridization (D FISH) in human sperm. Methods In situ hybridization of interphase nuclei in 12 normal sperms by two-color fluorescence in situ hybridization was performed. The number of hybridization signal particles in X, Y chromosome of spermatozoa was calculated. Results Under the microscope, the pBamX7 probe labeled with Biotin showed one green hybridization signal was X chromosome sperm (X spermatozoa) in the head of spermatozoa, and one red hybridization signal was Y chromosome sperm with Digoxigenin labeled pY3.4 probe (Y spermatozoa). Sperm with two fluorescence hybridization signals in the head of spermatozoon was abnormal chromosome number (such as XX, XY and YY spermatozoa); interphase nuclear background was blue with DAPI complex staining; 4 80 0 sperm nuclei, X sperm hybridization signal positive rate was 8.565%, Y sperm hybridization signal positive rate was 4 9.73%, a total of 240,000 sperm, the total of three kinds of diploid hybridization rate 0 .197%. Conclusion This method has the advantages of direct and easy distinguishing of fluorescence hybridization signal, large amount of analysis of sperm quantity in a short time and relatively simple and convenient experimental operation, accurate and credible results