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.

通过免疫Balb/c小鼠的急性巨核细胞白血病细胞系（M-MOK）细胞，我们制备了两种对CD 117（c-kit）具有特异性的单克隆抗体，MTK 1和MTK 2。我们发现用MTK 1和免疫磁珠以及CD 34抗体和磁珠处理骨髓单个核细胞能够纯化造血干细胞（CFU-GM和BUFU-E）。为了了解CD 117在白血病细胞和白血病细胞系中的阶段和谱系特异性表达，我们检测了白血病细胞和白血病细胞系。CD 117表达约70%的髓系白血病细胞。在白血病细胞体外适应过程中，巨核/红巨核细胞系CD 117的优势表达已被发现，但其机制尚不清楚，为了从另一个角度研究骨髓造血细胞，我们检测了CD 34阳性细胞上IL-2受体γ c链的表达。我们发现大约40%的CD 34阳性细胞表达γ c链。然后检测CD 34（+）γ c链（+）和CD 34（+）γ c链（-）细胞中CFU-GM和BFU-E的数量。CD 34（+）γ c链（-）细胞比CD 34（+）γ c链（+）细胞含有更多的BFU-E。我们尝试用电穿孔法将bcr/abl cDNA导入依赖GM-CSF的M-MOK细胞，作为基因转染造血细胞的初步实验。经G418筛选后的M-MOK细胞中存在Bcr/abl cDNA，用RT-PCR方法证实。然而，在bcr/abl cDNA的影响下，GM-CSF依赖性M-MOK细胞并没有转化为细胞因子非依赖性M-MOK细胞。为了将基因导入造血干细胞，似乎有许多关于载体和靶细胞的问题需要尽快解决。

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