荧光标记短串联重复序列复合扩增监测嵌合体的应用研究

Application of Short-tandem-repeat Amplification and Fluorescent-multiplex PCR for Chimerism Analysis

本研究采用荧光标记的多重PCR复合扩增短串联重复序列(STR)结合全自动毛细管电泳检测异基因造血干细胞移植后嵌合体水平,并探讨该方法动态监测对移植患者预后判断的作用。采集30例异基因造血干细胞移植患者及其供者移植前后骨髓或外周血的DNA,采用Profiler Plus试剂盒进行PCR扩增,产物经ABI310遗传分析仪进行毛细管电泳,通过供受者基因位点差异和峰面积进行嵌合体的定量检测。结果表明,29例患者在移植后28天形成完全供者嵌合体(complete chimerism,CC),1例形成混合嵌合体(mixed chimerism,MC)。在长期随访中,22例表现为持续完全供者嵌合体,8例混合嵌合体患者中7例原病复发,从完全供者嵌合体转为混合嵌合体。完全供者嵌合体组移植物抗宿主病(GVHD)的发生率明显高于混合嵌合体组。结论 :荧光标记多重复合扩增STR检测嵌合体具有快速、自动化高、可定量及灵敏度高等优点,动态定量监测嵌合体可用于移植动力学研究,对移植物早期植入或被排斥、疾病复发以及GVHD均有预警作用,对实施临床干预治疗有指导价值。

The purpose of this study was to detect chimerism status of patients received allogeneic hematopoietic stem cell transplantation by using short tandem repeat （STR） amplification and fluorescence labeling mulitplex polymerase chain reaction （PCR） combined with capillary electrophoresis, and to evaluate the prognostic value of monitoring chimerism status. DNA from peripheral blood or bone marrow of donors and recipients in different time were extracted, 10 different STR markers were co-amplified in a single reaction by using AmpFSTR Profiler Plus PCR amplification kits. Separation of the PCR products and fluorescence detection were performed by ABI PRISM 310 Genetic Analyzer with capillary electrophoresis. The Genescan and Genotype software were used for size calling and quantification of peak areas. The formula to calculate donor chimerism levels was based on the different ailelic distribution types between donor and recipient. The results showed that 29 patients obtained complete donor chimerism and one patient obtained mixed chimerism in 28 days after transplantation. 22 patients continued complete donor chimerism and 8 patients showed mixed chimerism after long time follow up. 7 patients showed disease relapse after turning mixed chimerism from complete donor chimerism. The incidence of GVHD was higher in group of full donor chimerism. It is concluded that STR fluorescent-multiplex PCR is a rapid, automatic and sensitive method for chimerism tests after hematopoietic stem cell transplantation, which is a valuable tool as a dynamic monitoring for chimerism status to predict graft failure, disease relapse and occurrence of GVHD, and provides a basis for early clinical intervention in patients with allo-HSCT.