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。
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.
出处
《中华医学遗传学杂志》
EI
CAS
CSCD
2003年第2期147-150,共4页
Chinese Journal of Medical Genetics
基金
国家"863"高技术项目 (2 0 0 2AA2 1 60 91 )~~
