MHC基因反义RNA导入造血干/祖细胞的研究  

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

作　　者：邓宇斌[1,2] 李树浓[1,2] 梁天文[1,2] 王斌[1,2] 

机构地区：[1]中山医科大学病理生理学教研室 [2]中山医科大学免疫学教研室

出　　处：《中国免疫学杂志》1999年第1期23-26,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金;广东省自然科学基金

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（×）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。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ＢａｍＨＩ酶解，回收ＨＬＡＤＲＢ１ｃＤＮＡ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Ｇ４１８３００μｇ／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。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(1×10 5 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 than that of control(45.0%)with inhibition rate of 38.2%.Conclusion:These results suggested that transduction of antisense RNA of HLA DR gene into cord blood hematopoietic stem cells can successfully inhibited the expression of HLA DR antigen.These would provide evidences for the prevention and treatment of transplantation immune reaction in clinic cord blood transplantation.

关 键 词：造血干 祖细胞 MHC-Ⅱ类基因 移植免疫应答 

分 类 号：R392.4[医药卫生—免疫学]