荧光标记复合扩增短串联重复序列基因分析骨髓移植物的植入

目的 为了准确地识别骨髓移植物的植入状态，探讨PCR扩增短串联重复序列（short tandem repeat,RCR－STR）在亲缘或无关供者骨髓移植的预后及白血病复发中的预警作用。方法 建立荧光标记PCR－STR等位基因分析技术，采用单克隆磁珠提取DNA，四色荧光标记，于移植前、移植后7天－6个月不等，采集供、受者血液（2例回顾性研究患者刮取口腔粘膜脱落细胞作为术前样本）DNA作PCR－STR分析。结果 ①12例患者骨髓移植后10例为完全供者型，其中同胞损髓8例，HLA全相合77例，半相合1例；无关供者损髓1例，HLA全相合，母亲供髓1例，HLA-Ⅰ全相合，HLA－Ⅱ半相合。10例受者术后出现Ⅰ度移植物抗宿主病（GVHD），STR均完全和持续表达供者基因型，追求观察3－25个月，预后良好。②12例中1例由嵌合型转变为完全供者型，系同胞损髓，HLA－Ⅰ半相合，HLA－Ⅱ全相合；术后第30天PCR－STR表达供、受者双方基因型，第60天完全转变为供者基因型，而自身的基因在外周血中消失，预后良好。③12例中1例为持续未植入，HLA半相合同胞捐髓，术后虽然有血液学和临床情况的改善，但移植后7，14，21天STR分析始终仅显示受者基因型，移植后4个月死亡。结论 基于分子水平的移位点PCR－STR分析是骨髓移植后供者植入的精确标志。研究表明，PCR－STR植入分析的准确性优于任何传统技术，对移植效果有预警作用。

Analysis of short tandem repeats with fluorescence labeling polymerase chain reaction to document engraft menit following allogeneic bone marrow transplantation

OBJECTIVE: In order to determine the status of engraftment following allogeneic bone marrow transplantation (allo-BMT) and predict the outcome, short tandem repeats (STR) were analysed by fluorescence labeling polymerase chain reaction (PCR). METHOD: Fluorescence labeling PCR detecting STR 9 loci was established. DNA was extracted by monoclonal magnetic beads from recipients peripheral blood nucleated cells and labeled with four color fluorescences before and from 7 days to six months after allogeneic bone marrow transplantation. RESULTS: 1. STR and clinical feature of 12 patients after allogeneic bone marrow transplantation were analysed. In the ten patients with full donor chimeras, 8 received marrow grafts from HLA identical sibling donors, 1 from HLA identical unrelated donor and 1 from HLA haploidentical mother. All of the 10 patients had durable engraftment and survived disease-freely for 3-25 months of follow-up. 2. One patient received a HLA-AB loci mismatched graft and his STR showed mixed chimeras at day 30, and converted into full donor chimeras at day 60 after transplantation. 3. One patient received HLA haploidentical sibling donor stem cells failed to be engrafted and died 4 months later. CONCLUSION: Analysis of multi-loci of STR after allo-BMT provided a real indication of engraftment. Mixed chimeras after transplantation suggested that donor lymphocytes or bone marrow cells be re-infused to enhance the conversion from mixed chimeras to full donor chimeras. For the documentation of engraftment, analysis of STR was better than any other available methods.