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

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锭反应法(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／祖细胞基因转导效率。

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

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.