STR-PCR分析嵌合体在同种异基因造血干细胞移植中的应用

Application of Chimerism Analysis to Allogeneic Hematopoietic Stem Cell Transplantation by STR-PCR

利用荧光标记的多重PCR扩增短串联重复序列(STR-PCR)结合毛细管电泳检测供者细胞嵌合率(DC),以探讨该方法的连续检测对异基因造血干细胞移植(allo-HSCT)后转归的预警作用,采集27例清髓性外周血干细胞移植患者移植前、移植后不同时段的外周血或骨髓,DNA样本用Profiler Plus和Cofiler Plus商品化试剂盒扩增后,用ABI310遗传分析仪进行毛细管电泳,确定基因位点及峰面积,根据基因型的差异选择嵌合率计算公式。结果表明:两种试剂盒测得的DC嵌合率一致;在27对中能区别出供受差别的STR位点,Profiler Plus为6.3(4-9)个,Cofiler Plus为4.9(2-6)个。26例患者均在移植后28天出现供者细胞,1例患者未出现供者细胞。14例患者DC100%,均获得持久植入,至今仍无白血病生存;另有9例患者出现不稳定混合嵌合(MC)状态(DC为0%-90.2%),其中5例为血液学复发。27例病人中有6例死亡。上述5例复发患者均在出现临床症状前发生DC量下降;供者细胞完全嵌合组移植物抗宿主病(GVHD)的发生率高于MC组。结论:动态检测DC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的发生均有预警作用,对早期实施临床干预治疗有重要的指导意义。

The aim of this study was to analyze chimerism, evaluate the status of engraftment and predict the outcome of allogeneic hematopoietic stem cell transplantation （allo-HSCT） by multiple short tandem repeat （STR） amplification using fluorescence labeling polymerase chain reaction （PCR） combined with capillary electrophoresis. Peripheral blood and bone marrow in 27 patients who received myeloablative allogenetic cell transplantation were collected before and after transplantation in different times. 10 and 7 different STR markers were co-amplified in a single reaction by using a commercial AmpF/STR Profiler Plus/Cofiler 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 soft ware were used for size calling and quantification of peak areas. The formula to calculate donor chimerism values was based on the different allelic distribution type between donor and recipient. The results showed that donor chimerism was similar by the two methods. The median number of informative alleles was 6. 3（4 -9） by Profiler Plus and 4.9 （2 - 6） by Cofiler Plus. The donor alleles appeared in 26 patients on day 28 post transplantation. One patient was not observed to appear donor alleles. 14 patients with 100% donor chimerism （DC） had stable engraftment and they still survive in free leukemia. 9 patients had unstable mixed chimerism （DC： 0% -90.2% ）, and 5 of them relapsed after allo-HSCT, 6 patients died. Decrease of donor chimerism appeared prior to graft rejection and disease relapse. The incidence of GVHD was higher in group of full donor chimerism. It is concluded that dynamic monitoring donor chimerism by STR-PCR in combination with all auto-capillary electrophoresis is a valuable tool for predicting graft rejection, disease relapse and occurrence of GVHD, and provides a basis for early clinical intervention in the patients received allo-HSCT.