一种基于单核苷酸多态性位点的定量检测异基因造血干细胞移植后嵌合率的新方法  

A novel single nucleotide polymorphism-based method for quantitative assessment of chimerism after allogeneic stem cell transplantation

作　　者：邵宇[1] 王健民[1] 龚胜兰[1] 蔡在龙[1] 章卫平[1] 宋献民[1] 王利平[1] 

机构地区：[1]第二军医大学附属长海医院血液科,上海200433

出　　处：《中华血液学杂志》2010年第2期92-96,共5页Chinese Journal of Hematology

基　　金：国家自然科学基金（34871100）;上海市卫生系统基金（08JC1406500）;第二军医大学博士创新基金;上海市生物医学重大科技攻关项目（05DZ19327）

摘　　要：目的建立一种单核苷酸多态性-聚合酶链反应（SNP—PCR）定量检测异基因造血干细胞移植后供受嵌合率的新方法，探讨其可行性、准确性及优越性。方法利用18个SNP位点筛选移植前每一对供、受者，采用实时定量PCR（RQ—PCR）对筛选出的差异位点行嵌合率定量分析，通过倍比稀释、模拟嵌合与微卫星重复序列-PCR（STR—PCR）、性染色体双色荧光原位杂交（XY．FISH）和融合基因的定量检测，比较、验证方法的准确性及敏感度。结果①利用内参质粒标准品扩增的17次标准曲线，平均斜率为-3．39，平均截距为39．97，相关系数均〉0．995，扩增效率接近理想水平；批内差及批间差分别为0．50％和1．10％，在可控范围；与模拟混合嵌合相关系数在0．99以上；可重复敏感度达0．01％。②40例移植患者中，95％以上可以筛选出供、受者差异SNP位点；SNP—PCR与STR．PCR结果吻合率达96．7％，与XY—FISH结果比较差异无统计学意义（P〉0．05）；SNP—PCR与特定白血病融合基因检测结果比较，完全嵌合（CC）标本均未检出肿瘤相关的融合基因，部分嵌合（MC）标本融合基因均为阳性。结论SNP—PCR检测嵌合率准确、敏感、易行，具有极高的临床应用前景，克服了STR—PCR竞争抑制及扩增平台期偏倚的缺点，可以替代其进行临床常规检测。Objective To develop a novel single nuclcotide polymorphism （SNP）-PCR based meth- od for quantitative detection of chimerism after allogeneic haemopoietic stem cell transplantation （allo-HSCT）, and to explore its feasibility, accuracy and superiority. Methods 18 SNP loci were screened to identify informative markers for detecting chimerism in each donor/recipient pair before transplantation. Then the chimerism rate of each informative marker was analyzed by real-time quantitative PCR （RQ-PCR）. The accuracy and sensitivity were verified by multiple proportion dilution and analogy chimerism compared with quantitative detection of short tandem repeat （STR）-PCR, fluorescence in situ hybridization （FISH） and fusion gene. ResultsThe average slope of the 17 time amplications of the internal control plasmid was - 3.39, the average intercept was 39.97, correlation coefficients were more than 0. 995, which was close to the theoretical level. The intraand interassay variability was 0.50% and 1.1% , respectively, which were both in the allowed ranges. A linear correlation with artificial mixed chimerism is above 0.99 and a sensitivity of 0.01% proved reproducible.①At least one informative marker could be found in over 95% of 40 donor/recipient pairs. The results of the chimerisms derived from SNP-PCR were consistent with that from STR-PCR （96.7%）, FISH and fusion gene analasis （P 〉 0.05）;the quantitative results of special fusion gene transcripts were negtive in complete chimerism samples, arid positive in inixed chimerism samples. Conclusions This new assay which overcome the PCR competition and plateau biases of STR-PCR provides an accurate ,reliable and rapid quantitative assessment of mixed chimerism after allo-transplantation. It is highly promising for of clinical application and may take the place of STR-PCR in the conventional chimerisim assessment.

关 键 词：造血干细胞移植 嵌合体 多态性 单核苷酸 聚合酶链反应 

分 类 号：R440[医药卫生—诊断学]