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

基本信息

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

项目摘要

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.

我们已经建立了具有未成熟髓样特征的人类白血病细胞系M-MOK。 M-MOK仅在人类胚胎肺衍生的成纤维细胞系Hel-O中生长。组织化学检查表明，过氧化物酶和道格酯酶染色的M-MOK为阴性。表面标记物谱为CD34（+），CD33（+），CD41（+），CD42B（+），HLA-DR（ - ）和糖蛋白（ - ）。我们研究了M-MOK细胞在HEL-O上的生长机制。核孔膜用于检查M-MOK和HEL-O之间的天气直接接触M-MOK细胞的生长。证明M-MOK能够通过无直接接触的Hel-O得出的可溶性因子（S）生长。收集Hel-O的培养上清液并用作条件培养基（CM）。补充20％胎牛血清和20％CM的RPMI-1640培养基可诱导M-MOK细胞的增殖，而无需进料细胞。我们使用此CM补充培养基维持M-MOK细胞长达1个月。为了了解HEL-O衍生的可溶性因子的性质，我们试图使用各种单克隆抗体（例如抗VLA4，抗CD11A，抗CD11a，anti-G-CSF，anti-GM-CSF，anti-GM-CSF），抗GM-CSF和抗IL-3抗原抑制M-MOK细胞在HEL-O上的生长。仅抗GM-CSF抗体抑制了M-MOK细胞的生长。通过在培养基中添加抗GM-CSF抗体，通过HEL-O衍生的条件培养基诱导M-MOK增殖。这些数据清楚地表明，支持MOK生长的HEL-O的可溶因子可能是GM-CSF。 HEL-O中mRNA的Northern印迹杂交分析证实了HEL-O细胞中GM-CSF的mRNA存在。