原代骨髓基质细胞层对逆转录病毒载体介导的造血干/祖细胞基因转导的支持效应  

The Supportive Effect of Primary Bone Marrow Stromal Cell Layers on Retroviral-mediated Transduction of Human Hematopoietic Stem/Progenitor Cells

作　　者：阳小卫[1] 岑建农[1] 王玮[1] 夏学鸣[1] 陈子兴[1] 

机构地区：[1]苏州大学附属第一医院,江苏省血液研究所苏州215006

出　　处：《中国实验血液学杂志》2002年第5期383-386,共4页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目编号39770 331

摘　　要：为探讨建立原代骨髓基质细胞层的方法并观察基质接触对造血干 /祖细胞 (HSC/HPC)基因转移的影响 ,采用Ficoll Hypaque分离成人骨髓单个核细胞 (MNC) ,用基质细胞培养液培养 ,传代 4次以建立原代骨髓基质细胞层。将经细胞因子预刺激的造血干 /祖细胞接种到经辐照处理的基质细胞层上 ,进行逆转录病毒介导的多药耐药基因 (mdr1 )转导 ,以半固体集落培养和聚合酶链反应法 (PCR)检测长春新碱 (VCR)抗性集落数和mdr1基因扩增片段以测定转导效率。结果表明 :原代骨髓基质细胞培养传代 4 - 6周形成混合贴壁细胞层 ,主要由成纤维细胞组成。集落法和PCR法检测显示基质接触能提高骨髓造血干 /祖细胞基因转导效率 2 .1 - 3 .3倍 ,对逆转录病毒介导的造血干 /祖细胞基因转导具有明显的支持效应。结论 :基质细胞接触联合细胞因子作用可提高逆转录病毒介导的造血干To elucidate the effect of established primary bone marrow stromal layers on the gene transduction of human hematopoietic stem/progenitor cells (HSC/HPC), mononuclear cells(MNC) from adult bone marrow were isolated by centrifugation on Ficoll Hypaque gradients and plated in stromal culture medium. The cells were incubated until passage 4 to establish primary stromal layers. The HSC/HPC prestimulated by cytokines were transduced by retroviral supernatant containing mdr1 gene in presence of irradiated stroma contact support. Transduced cells were plated in a colony forming unit assay with and without vincristine(VCR) to assess the efficiency of transduction. Individual colonies were also analyzed by polymerase chain reaction(PCR) for the presense of provirus. The results showed that the mixed adherent cell layers were formed when adult bone marrow stromal cells were incubated for four to six weeks, mainly being composed of fibroblasts. In the presence of stroma contact support, the average of gene transduction efficiency in marrow derived progenitors increased 2.1 to 3.3 folds measured by colony forming assay and/or PCR, significantly higher than those without support of stroma. It is concluded that the presence of bone marrow stroma support in combination with cytokine faciliates augmenting the extent of retroviral mediated gene transduction.

关 键 词：原代骨髓基质细胞 造血干细胞 造血祖细胞 基因转移 多药耐药基因 

分 类 号：Q78[生物学—分子生物学] R331.2[医药卫生—人体生理学]