Becker M, Nitsche A, Neumann C, Aumann J, Junghahn I, Fichtner I
Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13092 Berlin, Germany.
Br J Cancer. 2002 Nov 18;87(11):1328-35. doi: 10.1038/sj.bjc.6600573.
The sensitive detection of human cells in immunodeficient rodents is a prerequisite for the monitoring of micrometastasis of solid tumours, dissemination of leukaemic cells, or engraftment of haematological cells. We developed a universally applicable polymerase chain reaction method for the detection of a human-specific 850-bp fragment of the alpha-satellite DNA on human chromosome 17. The method allows the detection of one human cell in 10(6) murine cells and could be established as both, a conventional DNA polymerase chain reaction-assay for routine screening, and a quantitative real-time polymerase chain reaction-assay using TaqMan-methodology. It was applied to the following xenotransplantation systems in SCID and NOD/SCID mice: (1) In a limiting dilution assay, cells of the MDA-MB 435 breast carcinoma were injected into the mammary fat pad of NOD/SCID mice. It could be shown that 10 cells mouse(-1) were sufficient to induce a positive polymerase chain reaction signal in liver and lung tissue 30 days after transplantation as an indicator for micrometastasis. At this time a palpable tumour was not yet detectable in the mammary fat pad region. (2) Cells of a newly established human acute lymphatic leukaemia were administered intraperitoneally to SCID mice. These cells apparently disseminated and were detectable as early as day 50 in the peripheral blood of living mice, while the leukaemia manifestation was delayed by day 140. (3) In a transplantation experiment using mature human lymphocytes we wanted to standardise conditions for a successful survival of these cells in NOD/SCID mice. It was established that at least 5 x 10(7) cells given intravenously were necessary and that the mice had to be conditioned by 2 Gy body irradiation to get positive polymerase chain reaction bands in several organs. (4) Engraftment studies with blood stem cells originating from cytapheresis samples of tumour patients or from cord blood were undertaken in NOD/SCID mice in order to define conditions of successful engraftment and to use this model for further optimisation strategies. The polymerase chain reaction method presented allowed a reliable prediction of positive engraftment and agreed well with the results of immunohistochemical or FACS analysis. All together, the polymerase chain reaction method developed allows a sensitive and reliable detection of low numbers of human cells in immunodeficient hosts. In combination with real-time (TaqMan) technique it allows an exact quantification of human cells. As this method can be performed with accessible material of living animals, follow up studies for the monitoring of therapeutic interventions are possible in which the survival time of mice as evaluation criteria can be omitted.
在免疫缺陷啮齿动物中灵敏检测人类细胞是监测实体瘤微转移、白血病细胞播散或血液细胞植入的前提条件。我们开发了一种普遍适用的聚合酶链反应方法，用于检测人类17号染色体上α卫星DNA的一个850 bp的人类特异性片段。该方法能够在10⁶个鼠细胞中检测到一个人类细胞，并且既可以作为常规DNA聚合酶链反应检测方法用于常规筛查，也可以作为使用TaqMan技术的定量实时聚合酶链反应检测方法。它被应用于SCID和NOD/SCID小鼠的以下异种移植系统：(1)在有限稀释试验中，将MDA-MB 435乳腺癌细胞注射到NOD/SCID小鼠的乳腺脂肪垫中。结果表明，每只小鼠注射10个细胞就足以在移植后30天在肝脏和肺组织中诱导出阳性聚合酶链反应信号，作为微转移的指标。此时在乳腺脂肪垫区域尚未检测到可触及的肿瘤。(2)将新建立的人类急性淋巴细胞白血病细胞腹腔注射给SCID小鼠。这些细胞明显发生播散，早在第50天就能在存活小鼠的外周血中检测到，而白血病表现延迟至第140天。(3)在一项使用成熟人类淋巴细胞的移植实验中，我们想要标准化这些细胞在NOD/SCID小鼠中成功存活的条件。已确定静脉注射至少5×10⁷个细胞是必要的，并且小鼠必须接受2 Gy全身照射才能在多个器官中获得阳性聚合酶链反应条带。(4)在NOD/SCID小鼠中进行了源自肿瘤患者血细胞分离样本或脐血的造血干细胞植入研究，以确定成功植入的条件，并将该模型用于进一步的优化策略。所提出的聚合酶链反应方法能够可靠地预测阳性植入，并且与免疫组织化学或FACS分析结果吻合良好。总之，所开发的聚合酶链反应方法能够灵敏可靠地检测免疫缺陷宿主中少量的人类细胞。结合实时(TaqMan)技术，它能够对人类细胞进行精确量化。由于该方法可以用活体动物的可获取材料进行操作，因此可以进行后续研究以监测治疗干预，其中可以省略将小鼠存活时间作为评估标准。
