ESTABLISHMENT AND CHARACTERIZATION OF AN IMMATURE MYELOID CELL LINE, M-MOK, GROWN IN THE PRESENCE OF FIBROBLAST-FEEDER CELLS

在成纤维细胞饲养细胞存在下生长的未成熟骨髓细胞系 M-MOK 的建立和表征

• 批准号: 03454260
• 负责人: TSUCHIYA Shigeru
• 金额: $ 3.71万
• 依托单位: RESEARCH INSTITUTE FOR TUBERCULOSIS AND CANCER, TOHOKU UNIVERSITY (RITC,TU)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454260/
• 关键词: IMMATURE MYELOYD LEUKEMIA CELL LINE; FEEDER CELL DEPENDENCY; SOLUBLE FACTOR; GM-CSF; HEMATOPOIETIC STEM CELLS; 末熟骨髄性白血病細胞株; GMーCSF

We have established an human leukemia cell line, M-MOK, with immature myeloid features. M-MOK was grown only in the presence of human embryonic lung derived fibroblast cell line, HEL-O. Histochemical examination revealed that M-MOK was negative for peroxidase and dougle esterase stains. Surface marker profiles were CD34(+),CD33(+),CD41(+),CD42b(+),HLA-DR(-)and glycophorin (-). We investigated the mechanism of growth for M-MOK cells on HEL-O. Nucleopore membrane was used to examine weather direct contact between M-MOK and HEL-O was required for the growth of M-MOK cells. It was demonstrated that M-MOK was able to grow by soluble factor(s) derived from HEL-O without direct contact. Culture supernatant from HEL-O was harvested and used as conditioned medium(CM). RPMI-1640 medium supplemented with 20% fetal calf serum and 20% CM could induce proliferation of M-MOK cells without feeder cells. We maintained M-MOK cells using this CM supplemented medium as long as 1 month. In order to know the nature of the HEL-O derived soluble factors we tried to inhibit the growth of M-MOK cells on HEL-O using various monoclonal antibodies such as anti-VLA4,anti-CD11a, anti-G-CSF, anti-GM-CSF and anti-IL-3 antibodis. Only anti-GM-CSF antibody inhibited the growth of M-MOK cells. Induction of M-MOK proliferation by HEL-O derived conditioned medium was also blocked by the addition of anti-GM-CSF antibody to the culture medium. These data clearly indicated that the soluble factor from HEL-O which support the growth of M-MOK might be GM-CSF. Northern blot hybridization assay of mRNA from HEL-O confirmed the presence of mRNA for GM-CSF in HEL-O cells.

我们已经建立了一个人类白血病细胞系，M-MOK，具有未成熟的髓系特征。M-MOK仅在人胚胎肺源性成纤维细胞系HEL-O存在的情况下生长。组织化学检查显示M-MOK过氧化物酶和双酯酶染色阴性。表面标记谱为CD34（+）、CD33（+）、CD41（+）、CD42b（+）、HLA-DR（-）和糖蛋白（-）。我们研究了M-MOK细胞在hell - o上的生长机制。采用核孔膜检测M-MOK细胞生长是否需要与helo直接接触。结果表明，M-MOK可以在没有直接接触的情况下通过hell - o衍生的可溶性因子生长。收集hell - o培养上清液作为条件培养基（CM）。RPMI-1640培养基中添加20%胎牛血清和20% CM可诱导M-MOK细胞在无饲养细胞的情况下增殖。我们使用这种添加CM的培养基维持M-MOK细胞长达1个月。为了了解hell - o衍生可溶性因子的性质，我们尝试使用抗vla4、抗cd11a、抗g - csf、抗gm - csf和抗il -3抗体等多种单克隆抗体抑制hell - o上M-MOK细胞的生长。只有抗gm - csf抗体能抑制M-MOK细胞的生长。在hell - o衍生的条件培养基中加入抗gm - csf抗体也能阻断M-MOK的增殖。这些数据清楚地表明，hell - o中支持M-MOK生长的可溶性因子可能是GM-CSF。hell - o mRNA的Northern blot杂交实验证实了hell - o细胞中GM-CSF mRNA的存在。

项目成果

Masayoshi Itano et al.: "Establishment and charactcrization of an immmmature myelovd cell live,M-MOK,grown in the presence of fitroflasts-feeder-cells."

Masayoshi Itano 等人：“未成熟骨髓活细胞 M-MOK 的建立和表征，该细胞在 fitroflasts 饲养细胞存在下生长。”

Shigeru Tsuchiya: "Bーlineage phenotype of lymphoblastoch cells from patients with xーlinked Agammaglosulinemia" Tohoku J.Exp.Meal.163. 289-294 (1991)

Shigeru Tsuchiya：“来自 x 连锁无丙球蛋白血症患者的淋巴母细胞细胞的 B 谱系表型”Tohoku J.Exp.Meal.163 (1991)。

Masayoshi Minegishi et al.: "A Japanese family pedigree of patients with severe combined immmmunodeficiency clisease with X-linked in heritance" Jap.J.Heem.Genet.36. 137-142 (1991)

Masayoshi Minegishi 等人：“具有 X 连锁遗传的严重联合免疫缺陷病患者的日本家族谱系”Jap.J.Heem.Genet.36。

Masayoshi Itano et al.: "Mass Screening for neuroblastima in Miyagi Prefecture."

Masayoshi Itano 等人：“宫城县神经母细胞瘤的大规模筛查。”

Yuzo Jwoski et al.: "Subaoute paneneephalitis ossociated with chronie GVHD" Eur.J.Potholgy.

Yuzo Jwoski 等人：“与慢性 GVHD 相关的 Subaoute 全脑炎”Eur.J.Potholgy。