异基因造血干细胞移植后早期嵌合状态研究  

The chimerism in the early stage after allogeneic hematopoietic stem cell transplantation

作　　者：常伟[1] 孙汉英[1] 黄敏[1] 肖毅[1] 方慧[2] 周剑峰[1] 刘文励[1] 张义成[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院血液科,武汉430030 [2]武汉市公安局刑事侦查处

出　　处：《临床血液学杂志》2006年第6期347-349,共3页Journal of Clinical Hematology

摘　　要：目的:建立多重PCR扩增短串联重复序列(STR)结合毛细管电泳,定量检测异基因造血干细胞移植(Allo-HSCT)后受者体内供、受体来源血细胞嵌合率的方法及灵敏度。并探讨该方法的早期定量检测对Allo-HSCT后白血病早期复发进行干预的指导意义。方法:将2名健康成人静脉血分离单个核细胞,并按供、受体单个核细胞不同百分比制成模拟嵌合体标本并提取基因组DNA,用多重PCR扩增后,进行毛细管电泳,确定基因位点及相应峰面积。15例Allo-HSCT患者移植前采集供、受者外周血,移植后采集受者外周血,分别提取DNA,进行PCR扩增和毛细管电泳,确定STR基因位点及峰面积,计算混合样本中2单个样本DNA含量百分比及Al-lo-HSCT患者嵌合率。结果:①当混合样本中某一细胞比例为4%以上时,即可从电泳图中明确区分混合样本中该单个样本的基因型,且扩增前细胞百分率与扩增后两者峰面积比值呈直线相关;②所有患者在移植后2周内均出现供者来源的细胞,13例患者在术后30d内均达稳定嵌合。2例患者移植后出现不稳定混合嵌合状态或复发,经干预治疗后转为完全嵌合状态。结论:STR毛细管电泳PCR方法非常敏感,能应用于Allo-HSCT后早期嵌合体的定量检测。嵌合状态的早期定量检测对了解Allo-HSCT后的移植物的植入状态和指导早期干预治疗均有重要参考价值。Objective：To establish a quantitative assay of chimerism after Allo-HSCT by amplification of multiple short tandem repeat （STR） through fluorescence labeling polymerase chain reaction （PCR） combined with capillary electrophoresis, and to guide the intervention of the early leukemia relapse after the allogeneic hematopoietic stem cell transplantation. Method： Mononuclear cells were separated from blood samples of healthy unrelated donors or Allo HSCT donor/recipient pairs; nine STR markers were co-amplified in a single reaction by using a PCR amplification kit. The chimerism in Allo-HSCT was calculated on the basis of DNA percentage derived from the peak area of the electrophoresis of the patient samples. Result： （1）When a sample in the mixed proportion of the mononuclear cell of two samples was up to 4 G, the genotype of this specific sample of the mixture can be definitely distinguished, the mixed percentage before amplification and the ratio of their peak areas after amplification showed excellent linear correlation; （2）The donor-origin blood cells were detected in all Allo-HSCT patients within 14 days. One patient had unstable mixed chimerism on day 29 and another patient experienced overt leukemia relapse, both converted to complete donor chimerism following taper of cyclosporin A and/or donor lymphocyte infusion （DLI）. Conclusion：STR amplification by fluorescence labeling PCR combined with capillary electrophoresis is highly sensitive; Early quantitative monitoring of chimerism by STR-PCR is of valuable to verify the engraftment and leukemia relapse and to guide early intervention.

关 键 词：异基因造血干细胞移植 短片段串联重复 嵌合体 

分 类 号：R617[医药卫生—外科学]