巢式聚合酶链反应-序列特异引物法用于单卵裂球HLA-A位点分型研究  

HLA-A site genotyping on single blastomeres is studied by nest-PCR-SSP method

作　　者：徐炳森[1] 胡咏武[2] 黄学锋[1] 林金菊[1] 周颖[1] 叶碧绿[1] 徐力辛[1] 许康朴[3] 杨焕明[4] 

机构地区：[1]温州医学院附属第一医院生殖医学中心,325000 [2]温州医学院生命科学学院,325000 [3]美国康奈尔大学医学院 [4]中国科学院遗传与发育生物学研究所

出　　处：《中华医学遗传学杂志》2006年第2期156-160,共5页Chinese Journal of Medical Genetics

基　　金：温州医学院发展基金重大项目(2003001)~~

摘　　要：目的评价巢式聚合酶链反应．序列特异引物法（polymerase chain reaction-sequence specific primer，PCR-SSP）用于人类种植前胚胎的单个卵裂球HLA-A位点分型的准确性和可靠性。方法通过扩增HLA-A第2外显子评价单卵裂球巢式第1轮扩增的成功率，用巢式PCR-SSP法进行单卵裂球HLA-A分型。结果从10个家系的体外受精剩余胚胎中获得120个卵裂球，巢式第1轮扩增平均成功率为86．7％，其中前期家系F1-F5为78．2％，后期家系F6-F10为93．8％；对80个卵裂球进一步做SSP分型，其中11个卵裂球HLA-A第2外显子扩增失败，无分型结果，而69个卵裂球HLA-A第2外显子扩增成功，都获得分型结果。除6个卵裂球因亲本纯合无法判断是否发生缺失外，剩下63个卵裂球中，基因型与根据亲本预测的结果相符的有59个，占93．6％；显示缺少部分亲本等位基因的有3个，占4．8％；显示部分亲本等位基因重复的1个，占1．6％。结论在第1轮扩增成功的基础上，巢式PCR-SSP法用于单卵裂球HLA-A分型准确可靠，可用于临床上筛选与需要造血干细胞移植的患儿HLA型等同的胚胎。Objective To assess the accuracy and reliability of the nest-PCR-sequence specific primer（SSP） method in HLAoA site genotyping of single blastomeres retrieved from human pre-implantation embryos. Methods By nest PCR on HLA-A exon 2, the success rate of first-round amplification was estimated for single blastomeres. Based on the first-round amplification, the HLA-A genotype of every single blastomeres was analyzed by commercially available PCR-SSP kits. Results The amplification of HLA-A exon 2 were performed to 120 blasotmeres retrieved from /n v/tro fertillzation（IVF） surplus embryos donated by 10 couples. The average success rate of family 1-5 and 6-10 was 78.2% （ 43/55 ） and 93.8 % （61/65 ）, respectively. And 86.7 % （104/120） in total. Eighty blastomeres were further tested by nest-PCR-SSP, among which 11 blastomeres failed to HLA-A exon 2 amplification and then failed to genotyping while the other 69 blastomeres succeed in HLA-A exon 2 amplification and succeed in genotyping. Except for 6 blastomeres that were uncertain for allele lost because of parents＇ homozygosity, the left 63 blastomeres had accurate HLA genotyping. A- mong these 63 blastomeres, 59 blastomeres had genotypes confirmed from their parents （93.6%）, 3 blastomeres lost one of parents＇ alleles （4.8%）, and only one blastomere had two more than parents＇ alleles （ 1.6% ）. Conclusion The above research results indicated that based on the successful first round amplification of single blastomeres, nest-PCR-SSP strategy offers a convenient and reliable option for HLA genotyping on single blastomeres, which is a key process in preselecting HLA-identical sibling for allogeneic cord blood cell transplantation.

关 键 词：单卵裂球 种植前胚胎 人类白细胞抗原分型 

分 类 号：R714.8[医药卫生—妇产科学]