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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。
The cDNA fragment of HLA-DRBI gene was inserted reversely into the BamHI site of retroviral plasmid pZIP-neo SV (x) to construct a retroviral antisense RNA recombinant expression vector of HLA-DRB1 gene. The recombinant plasmid was introduced into PA317 cell. Immunomagnetic beads were used to separate and enrich cord blood CD34 ~ + cells. The cultured virus supernatant was infected cord blood CD34 ~ + cells, screened to obtain resistant clones and CFU-GM culture. The expression of the target gene of antisense RNA was confirmed by RT-PCR from resistant clones selected by G418 selection. The positive rate of HLA-DR antigen in transfected umbilical cord blood cells by flow cytometry (FACS) was 28% (control group: 45%), and the inhibition rate was 38.2%. The results showed that the recombinant MHC-Ⅱ antisense RNA introduced into cord blood cells could reduce the expression of HLA-DR antigen. Our results provide a new way to prevent graft-versus-host reaction in clinical cord blood transplantation using antisense nucleic acid technology.