MHC基因反义RNA导入造血干 祖细胞的研究

邓宇斌　李树浓　梁天文　王斌

　　中国图书分类号　R392.4
　　摘　要　目的：将HLA-DRB1基因cDNA片段反向插入逆转录病毒质粒ZIP-neoSV(×)Bam HI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。方法：用免疫磁珠法分离，富集CD34+脐血造血干/祖细胞。含编码人HLA-DRB1基因的pcDV1质粒，用Bam HI酶解，回收HLA-DRB1 cDNA片段，将cDNA片段反向插入pZIP-neoSV(×)逆转录病毒载体，经扩增、抽提、酶切鉴定，用脂质体将反义RNA重组体导入到PA317细胞，用含G418 300 μg/ml培养液筛选，获抗性克隆，流式细胞仪检测HLA-DR抗原阳性细胞数。结果：重组质粒转染PA317细胞后，其病毒滴度达1×105CFU/ml，脐血造血干/祖细胞经免疫磁珠富集后，CD34+细胞高达85.0%～90.0%，导入反义RNA的脐血干细胞，其HLA-DR抗原表达从导入前的45.0%降至28.0%，抑制率达38.2%，而导入空载体后从56.0%降至45.0%，差异不显著。结论：HLA-DR的反义RNA能导入脐血干细胞，降低HLA-DR抗原的表达。
　　关键词　造血干/祖细胞　基因转移　反义RNA　MHC-Ⅱ类基因

Antisense RNA of MHC gene were transducted into CD34+ cord blood stem/progenitor cells

DENG Yu-Bin,LI Shu-Nong,LIANG Tian-Wen et al.
Sun Yat-Sen University of Medical Sciences,Guangzhou 510089

　　　Abstract　Objective：To construct the retroviral vector encoding antisense RNA of HLA-DR gene and to transduct into packaging cell line PA317 by lipofectin.Methods:pcDV1 plasmid containing HLA-DRB1 gene (a gift from Dr long,NIH,USA) was distracted by alkaline lysation,and then digested by BamHI.pZIP-neoSV(X)vector digested by BamHI and T4 ligase were mixed with cDNA fragment at 18℃ for 16 h. Mononuclear cells of cord blood were incubated in CD34 monoclonal antibody (Milternyi Biotec,Germany) labeled micromagenetic balls for 12 min.After passing of the solution through the mini MACS column,the CD34 positive cells were collected.Results:A high titer(1×105 CFU/ml) helper-free virus producing cell PA317 was obtained .The CD34+ hematopoietic stem/progenitors cells were sorted and enriched by MACS.The percentage of CD34+ stem cell was increased to 85.0%～90.0%.The CD34+ cells were infected by the viral supernatant.The G418-resistant clone was demonstrated to be able to express the antisense RNA of HLA-DR gene by PCR technique. The expression rate of HLA -DR antigen of cord blood cell detected by FACS was 28.0%,which was apparently lower t

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