[摘要]目的 检测人endostatin(血管内皮抑制素)基因转染人脐带血CD34+ 造血干细胞后的表达和分泌情况。 方法 电穿孔法将Plncx.endo转染包装细胞PA317，G418筛选阳性克隆后用NIH3T3细胞测定病毒的滴度，RT.PCR法测定耐药的包装细胞中是否存在endostatin基因。取新鲜人脐带血40～80ml，用Ficoll分离出单个核细胞，再用免疫磁珠(MiniMACS)进一步分离出CD34+ 造血干细胞，流式细胞仪(FCM)检测CD34+ 造血干细胞的浓度和纯度。将病毒上清与人脐带血CD34+ 造血干细胞共同培养72h，应用RT.PCR及Western blot检测人endostatin的表达和分泌。 结果 Plncx.en-do质粒克隆扩增后，经酶切鉴定有endostatin基因存在。RT.PCR证实，转染人endostatin基因的PA317.endo、NIH3T3.endo细胞中存在人endostatin特异性片段，病毒滴度为1.3×10 5 cfu·ml-1 。FCM检测，CD34+ 干细胞在脐血中的初始含量<5%，经Mini.MACS分离纯化后纯度超过90%。从1×108 的界面细胞平均可以分离获得(5～20)×105 CD34+ 个干细胞。RT.PCR证实，脐带血CD34+ .endo造血干细胞基因组中含有人550bp endostatin特异性片段，Western blot分析显示人en-dostatin在转染细胞中获得稳定表达和分泌。 结论 逆转录病毒可以介导endostatin基因转导人脐带血CD34+ 造血干细胞并表达endostatin蛋白。

    [关键词] 逆转录病毒;基因;脐带血;造血干细胞;血管内皮抑制素

    Human endostatin gene transferred into human umbilical cord CD34+ hematopoietic stem cells

    HU Jie，JIANG Zao

    (Department of Oncology，Zhongda Hospital，Southeast University，Nanjing210009，China)

    Abstract:Objective To transfer human endostatin gene into umbilical cord blood CD34+ hematopoietic stem cells and to analyse its expression and excretion.Methods The recombinant plasmid plncx.endo was transferred into virus-packing cell PA317by electroporation.The clones of the cells transferred with human endostatin were se-lected by G418.Targeted NIH3T3cells were infected with the virus granules secreted from PA317and also selected by G418.The insertion and expression of endostatin gene in NIH3T3and PA317cells were analysed with RT.PCR.Human endostatin gene was transferred into CD34 ......