急性GVHD发生及未发生者移植物FOXP3 mRNA表达差异性探究.doc

急性GVHD发生及未发生者移植物FOXP3 mRNA表达差异性探究 　 作者：王志东　冯四洲　周世勇　王玫　周征　翟文静　韩明哲 【摘要】 　　本研究探讨移植物FOXP3 mRNA表达与HLA相合同胞供者异基因造血干细胞移植后急性移植物抗宿主病(aGVHD)的关系。对26例HLA相合同胞供者异基因造血干细胞移植患者移植物CD4+CD25+ 和 CD4+CD25high T 细胞、FOXP3 mRNA表达与aGVHD的关系进行了评价。用流式细胞术检测了脐血、正常人外周血、移植物的CD4+CD25+ 和 CD4+CD25highT 细胞比例；以β2-MG为内参照，实时定量RT-PCR检测了FOXP3 mRNA相对表达量，采用融解曲线和琼脂糖电泳鉴定PCR产物特异性。结果表明： 正常人外周血、动员的外周血干细胞（PBSC）和BM移植物CD4+CD25+ T细胞为连续的细胞群，CD4+CD25+ T 和 CD4+CD25highT 细胞比例分别为（48.5±16.3）%和（9.6±2.5）%，（42.1±14.7）%和（13.1±4.2）%，（43.4±9.6）%和（14.6±4.5）%。aGVHD组移植物CD4+CD25highT细胞比例和绝对值与无aGVHD组相比无显著差异（Pgt;0.05）。不同稀释倍数的FOXP3和β2-MG实时定量PCR相对扩增效率曲线斜率为0.0826；融解曲线分析表明均仅有单一峰，Tm值分别为86.5℃和 82.3℃；琼脂糖电泳显示都仅有单一扩增产物。PBSC移植中无aGVHD组FOXP3 mRNA相对表达量（中位数41.0×10-5）是有aGVHD组（中位数12.9×10-5）的318%，二者有显著性差异（P=0.03）。COX回归法分析排除aGVHD其他相关因素的影响。结论： CD25不是CD4+调控性T细胞可靠的细胞标志；应用SYBR Green I 实时定量RT-PCR方法可特异定量FOXP3 mRNA；aGVHD组移植物FOXP3 mRNA显著低于aGVHD未发生组。FOXP3是调控性T细胞可靠标志，可能是预测aGVHD的有用指标之一。 【关键词】 FOXP3；基因表达；急性移植物抗宿主病；异基因造血干细胞移植 　　Difference Between FOXP3 Gene Expressions in Donor Grafts with or without Acute Graft-versus-Host Disease 　　AbstractThe study was aimed to investigate the association of FOXP3 gene expression in donor grafts with acute graft-versus-host disease after HLA-identical sibling allogeneic hematopoietic stem cell transplantation. Twenty-six donor grafts (peripheral blood or bone marrow) and their respective clinical characteristics were evaluated. Flow cytometry analysis was performed to assess the percentage of CD4+CD25+ and CD4+CD25highT cells in cord blood, healthy controls’ peripheral blood and donor grafts. Relative transcripts of FOXP3 mRNA were determined by real-time quantitative reverse transcription -polymerase chain reaction with β2-MG as the internal control gene. The specificity of FOXP3 and β2-MG amplifications was confirmed by analyzing the dissociation curves and electrophoresis of the target amplicon. The results showed that the CD4+CD25+ T cells in peripheral blood, peripheral blood stem cell (PBSC) or BM grafts exhibited a continu