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

我们已经建立了一个具有未成熟髓系特征的人白血病细胞系M-MOK。M-MOK仅在人胚肺成纤维细胞系HEL-O存在下生长。组织化学检查显示M-MOK对过氧化物酶和双酯酶染色均为阴性。CD34(+)、CD33(+)、CD41(+)、CD42b(+)、人类白细胞抗原DR(-)和血糖蛋白(-)。我们研究了M-MOK细胞在HEL-O上的生长机制。用核孔膜检测M-MOK细胞是否需要与HEL-O直接接触才能生长。结果表明，M-MOK能够在HEL-O来源的可溶性因子(S)的作用下不直接接触生长。收集HEL-O细胞培养上清，作为条件培养液。添加20%胎牛血清和20%CM的RPMI-1640培养液可诱导无饲养层细胞的M-MOK细胞增殖。我们用这种添加CM的培养液维持M-MOK细胞长达1个月。为了了解HEL-O来源的可溶性因子的性质，我们尝试用抗VLA4、抗CD11a、抗G-CSF、抗GM-CSF和抗IL-3抗体等多种单抗来抑制M-MOK细胞在HEL-O上的生长。只有抗GM-CSF抗体能抑制M-MOK细胞的生长。加入抗GM-CSF抗体也可阻断HEL-O诱导的M-MOK增殖。提示HEL-O中支持M-MOK生长的可溶性因子可能是GM-CSF。对HEL-O来源的mRNA进行Northern印迹杂交分析，证实HEL-O细胞中存在GM-CSF的mRNA。

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.