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

CD117单克隆抗体MTK1纯化造血干细胞转染人类基因的初步研究

• 批准号: 06454295
• 负责人: TSUCHIYA Shigeru
• 金额: $ 4.29万
• 依托单位: INSTITUTE FOR DEVELOPMENT,AGING AND CANCER (IDAC) Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06454295/
• 关键词: HEMATOPOIETIC STEM CELLS; C-KIT; IL-2 RECEPTOR gamma CHAIN; ELECTROPORATION; GENE TRANSFECTION; BCR; ABL; IL-2セレプターγ鎖; CD34; 免疫磁気ビーズ; 白血病細胞株; bcr; abl; c-Kit

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.

通过对BALB/C小鼠的急性巨型巨细胞白血病细胞系（M-MOK）细胞进行免疫，我们产生了2种单克隆抗体，MTK1和MTK2，具有CD117（C-KIT）的特异性。我们发现用MTK1和免疫磁珠的骨髓单核细胞以及CD34抗体和珠子的处理能够净化造血干细胞（CFU-GM和BUFU-E）。在知道CD117的阶段和谱系特定表达中，我们检查了白血病细胞和白血病细胞系。 CD117表示约70％的髓样白血病细胞。在体外适应过程中，白血病细胞在带有巨型/红细胞核细胞谱系谱系细胞系上显性表达显性表达显性表达，但仍在研究中，但仍在研究该机制。表征骨髓造血细胞的表征不同的方式，我们以不同的方式检查了IL-2受体gamma C链在CD34阳性细胞上的表达。我们发现，大约40％的CD34阳性细胞表达了伽马链。然后，我们检查了CD34（+）γ链（+）和CD34（+）伽马链（ - ）细胞中CFU-GM和BFU-E的数量。与CD34（+）伽马链（+）细胞相比，CD34（+）伽马链（ - ）细胞包含更多的BFU-E。在这两个分数中，CFU-GM没有观察到的差异。我们试图通过电穿孔方法作为依赖性的M-MOK细胞中的BCR/ABL cDNA，作为将基因转染至造血细胞转染的初步实验。通过RT-PCR方法确认了G418选择后M-MOK细胞中BCR/ABL cDNA的存在。然而，在BCR/ABL cDNA的影响下，GM-CSF依赖的M-MOK细胞并未转化为独立的细胞因子。

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