【摘要】 目的　建立一种高度灵敏特异的间期荧光原位杂交 (interphasefluorescenceinsituhybridiza tion ,IFISH )技术 ,以检测人 /山羊嵌合模型中低比例的人细胞。方法　采用人特异Y染色体卫星DNA探针CEPY和 17号染色体卫星DNA探针p17H8对两头移植人男性造血干细胞 (hematopoieticstemcell,HSC)的人 /山羊嵌合模型、1头正常阴性山羊和 1名正常人男性的外周血进行IFISH分析。为评价FISH检测效率的正确性 ,将正常人男性与正常阴性山羊细胞按 1/10 0、1/5 0 0和 1/10 0 0混合进行IFISH分析。并确定一套信号计数规则 ,以保证方法稳定可靠。结果　人 /山羊混合细胞中人细胞阳性率与原比例相近。人细胞阳性率平均值为正常人男性 98 60 % (CEPY)和 10 0 0 0 % ( p17H8)、正常阴性山羊为 0、人 /山羊嵌合模型为 0 .2 3 % (CEPY)和 0 11% ( p17H8)。结果表明人 /山羊嵌合模型中有低比例的人细胞存在 ,且为男性细胞。结论　本研究建立的IFISH技术高度灵敏特异 ,能真实反映检测细胞的比例 ,为人 /山羊嵌合模型中低比例的人细胞检测提供了直观、灵敏、特异的途径。更多还原

【Abstract】 Objective To establish a high sensitive and specific method of interphase fluorescence in situ hybridization (IFISH) to detect the low frequency human cells in human/goat xenogeneic models. Methods Human specific Y chromosome satellite DNA CEPY and 17 chromosome satellite DNA p17H8 were used as probes for IFISH. The peripheral blood samples from 2 goats transplanted with human male hematopoietic stem cells (HSC), 1 normal negative goat and 1 normal man were analyzed. The actual FISH efficiency was confirmed by serial dilutions (1/100, 1/500 and 1/1000) of the cell mixture of normal man and normal negative goat. A set of signal scoring criteria was determined to guarantee the stability and reliability of the method. Results Positive cell (human cell) frequencies were consistent with the established frequencies for the human/goat cell mixture. The average frequencies of positive cells were 98 60%(CEPY) and 100 00% (p17H8) for normal man, 0 for normal negative goat, 0 23% (CEPY) and 0 11% (p17H8) for human/goat xenogeneic models. The results demonstrated that low frequency human cells (male cells confirmed by Y chromosome probe) existed in human/goat xenogeneic models. Conclusion The IFISH developed in this study is of high sensitivity and specificity and can identify the actual frequency of human cells, which offers a direct, sensitive and specific approach to the detection of low frequency human cells in human/goat xenogeneic models.更多还原