PURIFICATION OF HEMATOPOIETIC STEM CELLS BY A CD117 MONOCLONAL ANTIBODY,MTK1, IN ORDER TO TRANSFECT HUMAN GENES-A PRELIMINARY STUDY

通过对Balb/c小鼠免疫急性巨核母细胞白血病细胞系（M-MOK）细胞，我们产生了2种单克隆抗体，MTK1和MTK2，对CD117 （c-kit）具有特异性。我们发现用MTK1和免疫磁珠以及CD34抗体和磁珠治疗骨髓单核细胞能够纯化造血干细胞（CFU-GM和BUFU-E）。为了了解CD117的分期和谱系特异性表达，我们检测了白血病细胞和白血病细胞系。CD117表达约70%的髓系白血病细胞。在白血病细胞的体外适应过程中，我们注意到CD117在巨核/红巨核细胞系上的显性表达，但其机制仍在研究中。为了以不同的方式表征骨髓造血细胞，我们检测了IL-2受体γ - c链在CD34阳性细胞上的表达。我们发现大约40%的CD34阳性细胞表达γ - c链。然后我们检测了CD34 (+) γ c链（+）和CD34 (+) γ c链（-）细胞中CFU-GM和BFU-E的数量。与CD34 (+) γ c链（+）细胞相比，CD34 (+) γ c链（+）细胞含有更多的BFU-E。两组CFU-GM无显著差异。我们尝试用电穿孔法在GM-CSF依赖的M-MOK细胞中引入bcr/abl cDNA，作为基因转染造血细胞的初步实验。经G418筛选的M-MOK细胞经RT-PCR证实存在Bcr/abl cDNA。而依赖GM-CSF的M-MOK细胞在bcr/abl cDNA的影响下不转化为不依赖细胞因子的细胞。为了将基因引入造血干细胞，似乎有许多关于载体和靶细胞的问题需要尽快解决。

By immunizing an acute megakaryoblastic leukemia cell line (M-MOK) cells to Balb/c mice we have produced 2 monoclonal antibodies, MTK1 and MTK2, with the specificity to CD117 (c-kit). We found that treatment of bone marrow mononuclear cells with the MTK1 and immunomagnetic beads, as well as a CD34 antibody and the beads, was able to purify hematopoietic stem cells (CFU-GM and BUFU-E). In ordor to know stage and lineage specific expression of CD117 we examined leukemic cells and leukemia cell lines. CD117 expressed approximately 70% of myeloid leukemia cells. During in vitro adapation of leukemic cells dominant expression of CD117 on the cell lines with megakaryo/erythromegakaryocytic lineage was noticed, but the mechanism was still under investigation.To characterize bone marrow hematopoietic cells in a different way we examined the expression of the IL-2 receptor gamma c chains on CD34 positive cells. We found that approximately 40% of CD34 positive cells expressed gamma c chains. Then we examined the number of CFU-GM and BFU-E in CD34 (+) gamma c chain (+) and CD34 (+) gamma c chain (-) cells. CD34 (+) gamma c chain (-) cells contained more BFU-E as compared with those of CD34 (+) gamma c chain (+) cells. No differences were observed for CFU-GM in these 2 fractions.We tried to introduce bcr/abl cDNA in GM-CSF dependent M-MOK cells by an electroporation method as a preliminary experiment of transfection of genes to hematopoietic cells. Presence of Bcr/abl cDNA in M-MOK cells after selection by G418 was confirmed by a RT-PCR method. However, GM-CSF dependent M-MOK cells were not converted to cytokine independent one under the influence of bcr/abl cDNA.In order to introduce genes to hematopoietic stem cells there seemed to be many things concerning vectors and target cells which should be solved soon.