MHC-Ⅱ基因反义RNA导入脐血CD34^＋细胞抑制HLA-DR抗原的表达

将HLA-DRBI基因cDNA片段反向插入逆转录病毒质粒pZIP-neo SV(x)BamHI位点中,构建了HLA-DRB1基因的逆转录病毒反义RNA重组表达载体,用脂质体法导入PA317细胞。用免疫磁珠法分离、富集脐血CD34~ 细胞。将培养的病毒上清感染脐血CD34~ 细胞,筛选获得抗性克隆和进行CFU-GM的培养。用RT-PCR法从经G418选择产生的抗性克隆已证实有反义RNA目的基因的表达。流式细胞仪(FACS)检测转染的脐血细胞中HLA-DR抗原阳性率为28％(对照组为45％),其抑制率为38.2％。结果表明导入脐血细胞的MHC-Ⅱ类基因反义RNA重组体可降低其HLA-DR抗原的表达。本实验结果为用反义核酸技术在临床脐血移植中防止移植物抗宿主反应提供了新方法。

Transduction of antisense RNA of MHC-1 gene into cord blood CD34~ cells inhibits the expression of HLA-DR antigen

A retroviral vector encoding antisense RNA of HLA-DRB1 gene was constructed by molecular cloning technique and transducted into the packaging cell line PAS 17 by lipofection. The CD34 cells were sorted and enriched by magnetic cell sorting system (MACS). The percentage of CD34 cord blood cells was increased to 85%. The CD34 cells were infected by the mentioned viral supernatant. The HLA-DRB1 mRNA expression was detected in CD34 cells by RT-PCR technique. The expression rate of HLA-DR antigen on cord blood cells detected by FACS was 28%, which was significantly lower than that of the control (45 %) with a inhibition rate of 38.2%, P < 0.01. These results suggested that the introduction of antisense RNA of HLA-DR into cord blood hematopoietic stem/progenitor cells can effectively decrease the expression of HLA-DR antigen. These studies may provide a method for the prevention of graft versus host reaction (GVHR) in clinic cord blood transplantation.